Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing

ABSTRACT

CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region, which binds the target DNA by base-pairing, and a Cas9-binding region, to direct a Cas9 nuclease to a target DNA. Further disclosed are methods for increasing specificity of RNA-guided genome editing using CRISPR/Cas9 systems by using truncated guide RNAs (tru-gRNAs).

CLAIM OF PRIORITY

This application claims the benefit of U.S. Patent Application Ser. Nos. 61/799,647, filed on Mar. 15, 2013; 61/838,178, filed on Jun. 21, 2013; 61/838,148, filed on Jun. 21, 2013, and 61/921,007, filed on Dec. 26, 2013. The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. DP1 GM105378 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

BACKGROUND

Recent work has demonstrated that clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (Wiedenheft et al., Nature 482, 331-338 (2012); Horvath et al., Science 327, 167-170 (2010); Terns et al., Curr Opin Microbiol 14, 321-327 (2011)) can serve as the basis for performing genome editing in bacteria, yeast and human cells, as well as in vivo in whole organisms such as fruit flies, zebrafish and mice (Wang et al., Cell 153, 910-918 (2013); Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013); Gratz et al., Genetics 194(4):1029-35 (2013)). The Cas9 nuclease from S. pyogenes (hereafter simply Cas9) can be guided via base pair complementarity between the first 20 nucleotides of an engineered guide RNA (gRNA) and the complementary strand of a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG (Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013); Jinek et al., Science 337, 816-821 (2012)). Previous studies performed in vitro (Jinek et al., Science 337, 816-821 (2012)), in bacteria (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) and in human cells (Cong et al., Science 339, 819-823 (2013)) have shown that Cas9-mediated cleavage can, in some cases, be abolished by single mismatches at the gRNA/target site interface, particularly in the last 10-12 nucleotides (nts) located in the 3′ end of the 20 nt gRNA complementarity region.

SUMMARY

CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region (which binds the target DNA by base-pairing) and a Cas9-binding region, to direct a Cas9 nuclease to a target DNA (see FIG. 1). The nuclease can tolerate a number of mismatches (up to five, as shown herein) in the complementarity region and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using Cas9 or Cas9-based fusion proteins. In particular, provided are truncated guide RNAs (tru-gRNAs) that include a shortened target complementarity region (i.e., less than 20 nts, e.g., 17-19 or 17-18 nts of target complementarity, e.g., 17, 18 or 19 nts of target complementarity), and methods of using the same. As used herein, “17-18 or 17-19” includes 17, 18, or 19 nucleotides.

In one aspect, the invention provides a guide RNA molecule (e.g., a single guide RNA or a crRNA) having a target complementarity region of 17-18 or 17-19 nucleotides, e.g., the target complementarity region consists of 17-18 or 17-19 nucleotides, e.g., the target complementarity region consists of 17-18 or 17-19 nucleotides of consecutive target complementarity. In some embodiments, the guide RNA includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence. In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target sequence.

In another aspect, the invention provides a ribonucleic acid consisting of the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAAC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence (of 17-18 or 17-19 nucleotides) complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG (see, for example, the configuration in FIG. 1), and X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive

In another aspect, the invention provides DNA molecules encoding the ribonucleic acids described herein, and host cells harboring or expressing the ribonucleic acids or vectors.

In a further aspect, the invention provides methods for increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.

In yet another aspect, the invention provides methods for inducing a single or double-stranded break in a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase; and a guide RNA that includes a sequence consisting of 17 or 18 or 19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein.

Also provided herein are methods for modifying a target region of a double-stranded DNA molecule in a cell. The methods include expressing in or introducing into the cell: a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.

In some embodiments, the guide RNA is (i) a single guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, or (ii) a crRNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA.

In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive

In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ of any of the molecules described herein identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.

In some embodiments, one or more of the nucleotides of the RNA is modified, e.g., locked (2′-O-4′-C methylene bridge), is 5′-methylcytidine, is 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the target complementarity region X₁₇₋₁₈ or X₁₇₋₁₉. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X₁₇₋₁₈ or X₁₇₋₁₉ target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).

In an additional aspect, the invention provides methods for modifying a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell:

a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein. In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.

In another aspect, the invention provides methods for modifying, e.g., introducing a sequence specific break into, a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase, or a dCas9-heterologous functional domain fusion protein (dCas9-HFD);

a tracrRNA, e.g., comprising or consisting of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof; AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof; CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof; and a crRNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG; in some embodiments the crRNA has the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU.

In some embodiments the crRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) and the tracrRNA is GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8); the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404) and the tracrRNA is UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405); or the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) and the tracrRNA is AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406).

In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more U, e.g., 2 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, one or more of the nucleotides of the crRNA or tracrRNA is modified, e.g., locked (2′-O-4′-C methylene bridge), is 5′-methylcytidine, is 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the sequence X₁₇₋₁₈ or X₁₇₋₁₉. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X₁₇₋₁₈ or X₁₇₋₁₉ target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).

In some embodiments, the dCas9-heterologous functional domain fusion protein (dCas9-HFD) comprises a HFD that modifies gene expression, histones, or DNA, e.g., transcriptional activation domain, transcriptional repressors (e.g., silencers such as Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β), enzymes that modify the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins, e.g., TET1), or enzymes that modify histone subunit (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), or histone demethylases). In preferred embodiments, the heterologous functional domain is a transcriptional activation domain, e.g., a VP64 or NF-κB p65 transcriptional activation domain; an enzyme that catalyzes DNA demethylation, e.g., a TET protein family member or the catalytic domain from one of these family members; or histone modification (e.g., LSD1, histone methyltransferase, HDACs, or HATs) or a transcription silencing domain, e.g., from Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β; or a biological tether, e.g., MS2, CRISPR/Cas Subtype Ypest protein 4 (Csy4) or lambda N protein. dCas9-HFD are described in a U.S. Provisional Patent Application Ser. No. 61/799,647, Filed on Mar. 15, 2013, Attorney docket no. 00786-0882P02, U.S. Ser. No. 61/838,148, filed on Jun. 21, 2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.

In some embodiments, the methods described herein result in an indel mutation or sequence alteration in the selected target genomic sequence.

In some embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: Schematic illustrating a gRNA/Cas9 nuclease complex bound to its target DNA site. Scissors indicate approximate cleavage points of the Cas9 nuclease on the genomic DNA target site. Note the numbering of nucleotides on the guide RNA proceeds in an inverse fashion from 5′ to 3′.

FIG. 2A: Schematic illustrating a rationale for truncating the 5′ complementarity region of a gRNA. Thick grey lines=target DNA site, thin dark grey line structure=gRNA, black lines show base pairing (or lack thereof) between gRNA and target DNA site.

FIG. 2B: Schematic overview of the EGFP disruption assay. Repair of targeted Cas9-mediated double-stranded breaks in a single integrated EGFP-PEST reporter gene by error-prone NHEJ-mediated repair leads to frame-shift mutations that disrupt the coding sequence and associated loss of fluorescence in cells.

FIGS. 2C-F: Activities of RNA-guided nucleases (RGNs) harboring single guide RNAs (gRNAs) bearing (C) single mismatches, (D) adjacent double mismatches, (E) variably spaced double mismatches, and (F) increasing numbers of adjacent mismatches assayed on three different target sites in the EGFP reporter gene sequence. Mean activities of replicates are shown, normalized to the activity of a perfectly matched single gRNA. Error bars indicate standard errors of the mean. Positions mismatched in each single gRNA are highlighted in grey in the grid below. Sequences of the three EGFP target sites were as follows:

(SEQ ID NO: 9) EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO: 10) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO: 11) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG

FIG. 2G: Mismatches at the 5′ end of the gRNA make CRISPR/Cas more sensitive more 3′ mismatches. The gRNAs Watson-Crick base pair between the RNA & DNA with the exception of positions indicated with an “m” which are mismatched using the Watson-Crick transversion (i.e., EGFP Site#2 M18-19 is mismatched by changing the gRNA to its Watson-Crick partner at positions 18 & 19. Although positions near the 5′ of the gRNA are generally very well tolerated, matches in these positions are important for nuclease activity when other residues are mismatched. When all four positions are mismatched, nuclease activity is no longer detectable. This further demonstrates that matches at these 5′ position can help compensate for mismatches at other more 3′ positions. Note these experiments were performed with a non-codon optimized version of Cas9 which can show lower absolute levels of nuclease activity as compared to the codon optimized version.

FIG. 2H: Efficiency of Cas9 nuclease activities directed by gRNAs bearing variable length complementarity regions ranging from 15 to 25 nts in a human cell-based U2OS EGFP disruption assay. Expression of a gRNA from the U6 promoter requires the presence of a 5′ G and therefore it was only possible to evaluate gRNAs harboring certain lengths of complementarity to the target DNA site (15, 17, 19, 20, 21, 23, and 25 nts).

FIG. 3A: Efficiencies of EGFP disruption in human cells mediated by Cas9 and full-length or shortened gRNAs for four target sites in the EGFP reporter gene. Lengths of complementarity regions and corresponding target DNA sites are shown. Ctrl=control gRNA lacking a complementarity region.

FIG. 3B: Efficiencies of targeted indel mutations introduced at seven different human endogenous gene targets by matched standard RGNs (Cas9 and standard full-length gRNAs) and tru-RGNs (Cas9 and gRNAs bearing truncations in their 5′ complementarity regions). Lengths of gRNA complementarity regions and corresponding target DNA sites are shown. Indel frequencies were measured by T7EI assay. Ctrl=control gRNA lacking a complementarity region.

FIG. 3C: DNA sequences of indel mutations induced by RGNs using a tru-gRNA or a matched full-length gRNA targeted to the EMX1 site. The portion of the target DNA site that interacts with the gRNA complementarity region is highlighted in grey with the first base of the PAM sequence shown in lowercase. Deletions are indicated by dashes highlighted in grey and insertions by italicized letters highlighted in grey. The net number of bases deleted or inserted and the number of times each sequence was isolated are shown to the right.

FIG. 3D: Efficiencies of precise HDR/ssODN-mediated alterations introduced at two endogenous human genes by matched standard and tru-RGNs. % HDR was measured using a BamHI restriction digest assay (see the Experimental Procedures for Example 2). Control gRNA=empty U6 promoter vector.

FIG. 3E: U2OS.EGFP cells were transfected with variable amounts of full-length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) together with a fixed amount of Cas9 expression plasmid and then assayed for percentage of cells with decreased EGFP expression. Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained with tru-gRNA matches closely with data from experiments performed with full-length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites.

FIG. 3F: U2OS.EGFP cells were transfected with variable amount of Cas9 expression plasmid together with fixed amounts of full-length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) for each target (amounts determined for each tru-gRNA from the experiments of FIG. 3E). Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained with tru-gRNA matches closely with data from experiments performed with full-length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites. The results of these titrations determined the concentrations of plasmids used in the EGFP disruption assays performed in Examples 1 and 2.

FIG. 4A: Schematic illustrating locations of VEGFA sites 1 and 4 targeted by gRNAs for paired double nicks. Target sites for the full-length gRNAs are underlined with the first base in the PAM sequence shown in lowercase. Location of the BamHI restriction site inserted by HDR with a ssODN donor is shown.

FIG. 4B: A tru-gRNA can be used with a paired nickase strategy to efficiently induce indel mutations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases. Control gRNA used is one lacking a complementarity region.

FIG. 4C: A tru-gRNA can be used with a paired nickase strategy to efficiently induce precise HDR/ssODN-mediated sequence alterations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases with an ssODN donor template. Control gRNA used is one lacking a complementarity region.

FIG. 5A: Activities of RGNs targeted to three sites in EGFP using full-length (top) or tru-gRNAs (bottom) with single mismatches at each position (except at the 5′-most base which must remain a G for efficient expression from the U6 promoter). Grey boxes in the grid below represent positions of the Watson-Crick transversion mismatches. Empty gRNA control used is a gRNA lacking a complementarity region. RGN activities were measured using the EGFP disruption assay and values shown represent the percentage of EGFP-negative observed relative to an RGN using a perfectly matched gRNA. Experiments were performed in duplicate and means with error bars representing standard errors of the mean are shown.

FIG. 5B: Activities of RGNs targeted to three sites in EGFP using full-length (top) or tru-gRNAs (bottom) with adjacent double mismatches at each position (except at the 5′-most base which must remain a G for efficient expression from the U6 promoter). Data presented as in 5A.

FIG. 6A: Absolute frequencies of on- and off-target indel mutations induced by RGNs targeted to three different endogenous human gene sites as measured by deep sequencing. Indel frequencies are shown for the three target sites from cells in which targeted RGNs with a full-length gRNA, a tru-gRNA, or a control gRNA lacking a complementarity region were expressed. Absolute counts of indel mutations used to make these graphs can be found in Table 3B.

FIG. 6B: Fold-improvements in off-target site specificities of three tru-RGNs. Values shown represent the ratio of on/off-target activities of tru-RGNs to on/off-target activities of standard RGNs for the off-target sites shown, calculated using the data from (A) and Table 3B. For the sites marked with an asterisk (*), no indels were observed with the tru-RGN and therefore the values shown represent conservative statistical estimates for the fold-improvements in specificities for these off-target sites (see Results and Experimental Procedures).

FIG. 6C, top: Comparison of the on-target and an off-target site identified by T7EI assay for the tru-RGN targeted to VEGFA site 1 (more were identified by deep sequencing). Note that the full-length gRNA is mismatched to the two nucleotides at the 5′ end of the target site and that these are the two nucleotides not present in the tru-gRNA target site. Mismatches in the off-target site relative to the on-target are highlighted in bold underlined text. Mismatches between the gRNAs and the off-target site are shown with X's.

FIG. 6C, bottom: Indel mutation frequencies induced in the off-target site by RGNs bearing full-length or truncated gRNAs. Indel mutation frequencies were determined by T7EI assay. Note that the off-target site in this figure is one that we had examined previously for indel mutations induced by the standard RGN targeted to VEGFA site 1 and designated as site OT1-30 in that earlier study (Example 1 and Fu et al., Nat Biotechnol. 31(9):822-6 (2013)). It is likely that we did not identify off-target mutations at this site in our previous experiments because the frequency of indel mutations appears to be at the reliable detection limit of the T7EI assay (2-5%).

FIGS. 7A-D: DNA sequences of indel mutations induced by RGNs using tru-gRNAs or matched full-length gRNAs targeted to VEGFA sites 1 and 3. Sequences depicted as in FIG. 3C.

FIG. 7E. Indel mutation frequencies induced by tru-gRNAs bearing a mismatched 5′ G nucleotide. Indel mutation frequencies in human U2OS.EGFP cells induced by Cas9 directed by tru-gRNAs bearing 17, 18 or 20 nt complementarity regions for VEGFA sites 1 and 3 and EMX1 site 1 are shown. Three of these gRNAs contain a mismatched 5′ G (indicated by positions marked in bold text). Bars indicate results from experiments using full-length gRNA (20 nt), tru-gRNA (17 or 18 nt), and tru-gRNA with a mismatched 5′ G nucleotide (17 or 18 nt with boldface T at 5′ end). (Note that no activity was detectable for the mismatched tru-gRNA to EMX1 site 1.)

FIGS. 8A-C: Sequences of off-target indel mutations induced by RGNs in human U2OS.EGFP cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey.

FIGS. 9A-C: Sequences of off-target indel mutations induced by RGNs in human HEK293 cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey. *Yielded a large number of single by indels.

DETAILED DESCRIPTION

CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Although Marraffini and colleagues (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) recently performed a systematic investigation of Cas9 RGN specificity in bacteria, the specificities of RGNs in human cells have not been extensively defined. Understanding the scope of RGN-mediated off-target effects in human and other eukaryotic cells will be critically essential if these nucleases are to be used widely for research and therapeutic applications. The present inventors have used a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs. Single and double mismatches were tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. Off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells were readily detected by examination of partially mismatched sites. The off-target sites identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Thus RGNs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

The results described herein reveal that predicting the specificity profile of any given RGN is neither simple nor straightforward. The EGFP reporter assay experiments show that single and double mismatches can have variable effects on RGN activity in human cells that do not strictly depend upon their position(s) within the target site. For example, consistent with previously published reports, alterations in the 3′ half of the sgRNA/DNA interface generally have greater effects than those in the 5′ half (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al., Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)); however, single and double mutations in the 3′ end sometimes also appear to be well tolerated whereas double mutations in the 5′ end can greatly diminish activities. In addition, the magnitude of these effects for mismatches at any given position(s) appears to be site-dependent. Comprehensive profiling of a large series of RGNs with testing of all possible nucleotide substitutions (beyond the Watson-Crick transversions used in our EGFP reporter experiments) may help provide additional insights into the range of potential off-targets. In this regard, the recently described bacterial cell-based method of Marraffini and colleagues (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) or the in vitro, combinatorial library-based cleavage site-selection methodologies previously applied to ZFNs by Liu and colleagues (Pattanayak et al., Nat Methods 8, 765-770 (2011)) might be useful for generating larger sets of RGN specificity profiles.

Despite these challenges in comprehensively predicting RGN specificities, it was possible to identify bona fide off-targets of RGNs by examining a subset of genomic sites that differed from the on-target site by one to five mismatches. Notably, under conditions of these experiments, the frequencies of RGN-induced mutations at many of these off-target sites were similar to (or higher than) those observed at the intended on-target site, enabling the detection of mutations at these sites using the T7EI assay (which, as performed in our laboratory, has a reliable detection limit of ˜2 to 5% mutation frequency). Because these mutation rates were very high, it was possible to avoid using deep sequencing methods previously required to detect much lower frequency ZFN- and TALEN-induced off-target alterations (Pattanayak et al., Nat Methods 8, 765-770 (2011); Perez et al., Nat Biotechnol 26, 808-816 (2008); Gabriel et al., Nat Biotechnol 29, 816-823 (2011); Hockemeyer et al., Nat Biotechnol 29, 731-734 (2011)). Analysis of RGN off-target mutagenesis in human cells also confirmed the difficulties of predicting RGN specificities—not all single and double mismatched off-target sites show evidence of mutation whereas some sites with as many as five mismatches can also show alterations. Furthermore, the bona fide off-target sites identified do not exhibit any obvious bias toward transition or transversion differences relative to the intended target sequence (Table E; grey highlighted rows).

Although off-target sites were seen for a number of RGNs, identification of these sites was neither comprehensive nor genome-wide in scale. For the six RGNs studied, only a very small subset of the much larger total number of potential off-target sequences in the human genome (sites that differ by three to six nucleotides from the intended target site; compare Tables E and C) was examined. Although examining such large numbers of loci for off-target mutations by T7EI assay is neither a practical nor a cost-effective strategy, the use of high-throughput sequencing in future studies might enable the interrogation of larger numbers of candidate off-target sites and provide a more sensitive method for detecting bona fide off-target mutations. For example, such an approach might enable the unveiling of additional off-target sites for the two RGNs for which we failed to uncover any off-target mutations. In addition, an improved understanding both of RGN specificities and of any epigenomic factors (e.g., DNA methylation and chromatin status) that may influence RGN activities in cells might also reduce the number of potential sites that need to be examined and thereby make genome-wide assessments of RGN off-targets more practical and affordable.

As described herein, a number of strategies can be used to minimize the frequencies of genomic off-target mutations. For example, the specific choice of RGN target site can be optimized; given that off-target sites that differ at up to five positions from the intended target site can be efficiently mutated by RGNs, choosing target sites with minimal numbers of off-target sites as judged by mismatch counting seems unlikely to be effective; thousands of potential off-target sites that differ by four or five positions within the 20 bp RNA:DNA complementarity region will typically exist for any given RGN targeted to a sequence in the human genome (see, for example, Table C). It is also possible that the nucleotide content of the gRNA complementarity region might influence the range of potential off-target effects. For example, high GC-content has been shown to stabilize RNA:DNA hybrids (Sugimoto et al., Biochemistry 34, 11211-11216 (1995)) and therefore might also be expected to make gRNA/genomic DNA hybridization more stable and more tolerant to mismatches. Additional experiments with larger numbers of gRNAs will be needed to assess if and how these two parameters (numbers of mismatched sites in the genome and stability of the RNA:DNA hybrid) influence the genome-wide specificities of RGNs. However, it is important to note that even if such predictive parameters can be defined, the effect of implementing such guidelines would be to further restrict the targeting range of RGNs.

One potential general strategy for reducing RGN-induced off-target effects might be to reduce the concentrations of gRNA and Cas9 nuclease expressed in the cell. This idea was tested using the RGNs for VEGFA target sites 2 and 3 in U2OS.EGFP cells; transfecting less sgRNA- and Cas9-expressing plasmid decreased the mutation rate at the on-target site but did not appreciably change the relative rates of off-target mutations (Tables 2A and 2B). Consistent with this, high-level off-target mutagenesis rates were also observed in two other human cell types (HEK293 and K562 cells) even though the absolute rates of on-target mutagenesis are lower than in U2OS.EGFP cells. Thus, reducing expression levels of gRNA and Cas9 in cells is not likely to provide a solution for reducing off-target effects. Furthermore, these results also suggest that the high rates of off-target mutagenesis observed in human cells are not caused by overexpression of gRNA and/or Cas9.

TABLE 2A Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and single gRNA-expressing plasmids for the RGN targeted to VEGFA Target Site 2 12.5 ng gRNA/50 250 ng gRNA/750 ng ng Cas9 SEQ Cas9 Mean indel ID Mean indel frequency frequency Site Sequence NO: (%) ± SEM (%) ± SEM T2 (On-target) GACCCCCTCCACCCCGCCTCCGG 12 50.2 ± 4.9 25.4 ± 4.8 OT2-1 GACCCCC C CCACCCCGCC C CCGG 13 14.4 ± 3.4  4.2 ± 0.2 OT2-2 G GG CCCCTCCACCCCGCCTCTGG 14 20.0 ± 6.2  9.8 ± 1.1 OT2-6 CTA CCCCTCCACCCCGCCTCCGG 15  8.2 ± 1.4  6.0 ± 0.5 OT2-9 G C CCCC AC CCACCCCGCCTCTGG 16 50.7 ± 5.6 16.4 ± 2.1 OT2-15 T ACCCCC CA CACCCCGCCTCTGG 17  9.7 ± 4.5  2.1 ± 0.0 OT2-17 ACA CCCC C CCACCCCGCCTCAGG 18 14.0 ± 2.8  7.1 ± 0.0 OT2-19 ATT CCCC C CCACCCCGCCTCAGG 19 17.0 ± 3.3  9.2 ± 0.4 OT2-20 CC CC A CC C CCACCCCGCCTCAGG 20  6.1 ± 1.3 N.D. OT2-23 CG CCC T C C CCACCCCGCCTCCGG 21 44.4 ± 6.7 35.1 ± 1.8 OT2-24 CT CCCC AC CCACCCCGCCTCAGG 22 62.8 ± 5.0 44.1 ± 4.5 OT2-29 TG CCCC TC CCACCCCGCCTCTGG 23 13.8 ± 5.2  5.0 ± 0.2 OT2-34 AGG CCCC CA CACCCCGCCTCAGG 24  2.8 ± 1.5 N.D. Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods. OT = Off-target sites, numbered as in Table 1 and Table B. Mismatches from the on-target site (within the 20 bpregion to which the gRNA hybridizes) are highlighted as bold, underlined text. N.D. = none detected

TABLE 2B Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and single gRNA-expressing plasmids for the RGN targeted to VEGFA Target Site 3 250 ng gRNA/750 ng Cas9 SEQ Mean indel 12.5 ng gRNA/250 ng Cas9 ID frequency Mean indel frequency Site Sequence NO: (%) ± SEM (%) ± SEM T3 (On-target) GGTGAGTGAGTGTGTGCGTGTGG 25 49.4 ± 3.8 33.0 ± 3.7 OT3-1 GGTGAGTGAGTGTGTG T GTGAGG 26  7.4 ± 3.4 N.D. OT3-2 A GTGAGTGAGTGTGTG T GTGGGG 27 24.3 ± 9.2  9.8 ± 4.2 OT3-4 G C TGAGTGAGTGT A TGCGTGTGG 28  20.9 ± 11.8  4.2 ± 1.2 OT3-9 GGTGAGTGAGTG C GTGCG G GTGG 29  3.2 ± 0.3 N.D. OT3-17 G T TGAGTGA A TGTGTGCGTGAGG 30  2.9 ± 0.2 N.D. OT3-18 T GTG G GTGAGTGTGTGCGTGAGG 31 13.4 ± 4.2  4.9 ± 0.0 OT3-20 A G A GAGTGAGTGTGTGC A TGAGG 32 16.7 ± 3.5  7.9 ± 2.4 Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods. OT = Off-target sites, numbered as in Table 1 and Table B. N.D. = none detected

The finding that significant off-target mutagenesis can be induced by RGNs in three different human cell types has important implications for broader use of this genome-editing platform. For research applications, the potentially confounding effects of high frequency off-target mutations will need to be considered, particularly for experiments involving either cultured cells or organisms with slow generation times for which the outcrossing of undesired alterations would be challenging. One way to control for such effects might be to utilize multiple RGNs targeted to different DNA sequences to induce the same genomic alteration because off-target effects are not random but instead related to the targeted site. However, for therapeutic applications, these findings clearly indicate that the specificities of RGNs will need to be carefully defined and/or improved if these nucleases are to be used safely in the longer term for treatment of human diseases.

Methods for Improving Specificity

As shown herein, CRISPR-Cas RNA-guided nucleases based on the S. pyogenes Cas9 protein can have significant off-target mutagenic effects that are comparable to or higher than the intended on-target activity (Example 1). Such off-target effects can be problematic for research and in particular for potential therapeutic applications. Therefore, methods for improving the specificity of CRISPR-Cas RNA guided nucleases (RGNs) are needed.

As described in Example 1, Cas9 RGNs can induce high-frequency indel mutations at off-target sites in human cells (see also Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). These undesired alterations can occur at genomic sequences that differ by as many as five mismatches from the intended on-target site (see Example 1). In addition, although mismatches at the 5′ end of the gRNA complementarity region are generally better tolerated than those at the 3′ end, these associations are not absolute and show site-to-site-dependence (see Example 1 and Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). As a result, computational methods that rely on the number and/or positions of mismatches currently have limited predictive value for identifying bona fide off-target sites. Therefore, methods for reducing the frequencies of off-target mutations remain an important priority if RNA-guided nucleases are to be used for research and therapeutic applications.

Truncated Guide RNAs (Tru-gRNAs) Achieve Greater Specificity

Guide RNAs generally speaking come in two different systems: System 1, which uses separate crRNA and tracrRNAs that function together to guide cleavage by Cas9, and System 2, which uses a chimeric crRNA-tracrRNA hybrid that combines the two separate guide RNAs in a single system (referred to as a single guide RNA or sgRNA, see also Jinek et al., Science 2012; 337:816-821). The tracrRNA can be variably truncated and a range of lengths has been shown to function in both the separate system (system 1) and the chimeric gRNA system (system 2). For example, in some embodiments, tracrRNA may be truncated from its 3′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In some embodiments, the tracrRNA molecule may be truncated from its 5′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the tracrRNA molecule may be truncated from both the 5′ and 3′ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts on the 5′ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3′ end. See, e.g., Jinek et al., Science 2012; 337:816-821; Mali et al., Science. 2013 Feb. 15; 339(6121):823-6; Cong et al., Science. 2013 Feb. 15; 339(6121):819-23; and Hwang and Fu et al., Nat Biotechnol. 2013 March; 31(3):227-9; Jinek et al., Elife 2, e00471 (2013)). For System 2, generally the longer length chimeric gRNAs have shown greater on-target activity but the relative specificities of the various length gRNAs currently remain undefined and therefore it may be desirable in certain instances to use shorter gRNAs. In some embodiments, the gRNAs are complementary to a region that is within about 100-800 bp upstream of the transcription start site, e.g., is within about 500 bp upstream of the transcription start site, includes the transcription start site, or within about 100-800 bp, e.g., within about 500 bp, downstream of the transcription start site. In some embodiments, vectors (e.g., plasmids) encoding more than one gRNA are used, e.g., plasmids encoding, 2, 3, 4, 5, or more gRNAs directed to different sites in the same region of the target gene.

The present application describes a strategy for improving RGN specificity based on the seemingly counterintuitive idea of shortening, rather than lengthening, the gRNA complementarity region. These shorter gRNAs can induce various types of Cas9-mediated on-target genome editing events with efficiencies comparable to (or, in some cases, higher than) full-length gRNAs at multiple sites in a single integrated EGFP reporter gene and in endogenous human genes. In addition, RGNs using these shortened gRNAs exhibit increased sensitivity to small numbers of mismatches at the gRNA-target DNA interface. Most importantly, use of shortened gRNAs substantially reduces the rates of genomic off-target effects in human cells, yielding improvements of specificity as high as 5000-fold or more at these sites. Thus, this shortened gRNA strategy provides a highly effective approach for reducing off-target effects without compromising on-target activity and without the need for expression of a second, potentially mutagenic gRNA. This approach can be implemented on its own or in conjunction with other strategies such as the paired nickase method to reduce the off-target effects of RGNs in human cells.

Thus, one method to enhance specificity of CRISPR/Cas nucleases shortens the length of the guide RNA (gRNA) species used to direct nuclease specificity. Cas9 nuclease can be guided to specific 17-18 nt genomic targets bearing an additional proximal protospacer adjacent motif (PAM), e.g., of sequence NGG, using a guide RNA, e.g., a single gRNA or a crRNA (paired with a tracrRNA), bearing 17 or 18 nts at its 5′ end that are complementary to the complementary strand of the genomic DNA target site (FIG. 1).

Although one might expect that increasing the length of the gRNA complementarity region would improve specificity, the present inventors (Hwang et al., PLoS One. 2013 Jul. 9; 8 (7):e68708) and others (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9) have previously observed that lengthening the target site complementarity region at the 5′ end of the gRNA actually makes it function less efficiently at the on-target site.

By contrast, experiments in Example 1 showed that gRNAs bearing multiple mismatches within a standard length 5′ complementarity targeting region could still induce robust Cas9-mediated cleavage of their target sites. Thus, it was possible that truncated gRNAs lacking these 5′-end nucleotides might show activities comparable to their full-length counterparts (FIG. 2A). It was further speculated that these 5′ nucleotides might normally compensate for mismatches at other positions along the gRNA-target DNA interface and therefore predicted that shorter gRNAs might be more sensitive to mismatches and thus induce lower levels of off-target mutations (FIG. 2A).

Decreasing the length of the DNA sequence targeted might also decrease the stability of the gRNA:DNA hybrid, making it less tolerant of mismatches and thereby making the targeting more specific. That is, truncating the gRNA sequence to recognize a shorter DNA target might actually result in a RNA-guided nuclease that is less tolerant to even single nucleotide mismatches and is therefore more specific and has fewer unintended off-target effects.

This strategy for shortening the gRNA complementarity region could potentially be used with RNA guided proteins other than S. pyogenes Cas9 including other Cas proteins from bacteria or archaea as well as Cas9 variants that nick a single strand of DNA or have no-nuclease activity such as a dCas9 bearing catalytic inactivating mutations in one or both nuclease domains. This strategy can be applied to systems that utilize a single gRNA as well as those that use dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems).

Thus, described herein is a single guide RNA comprising a crRNA fused to a normally trans-encoded tracrRNA, e.g., a single Cas9 guide RNA as described in Mali et al., Science 2013 Feb. 15; 339(6121):823-6, but with a sequence at the 5′ end that is complementary to fewer than 20 nucleotides (nts), e.g., 19, 18, or 17 nts, preferably 17 or 18 nts, of the complementary strand to a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG. In some embodiments, the shortened Cas9 guide RNA consists of the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAAC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is the nucleotide sequence complementary to 17-18 or 17-19 consecutive nucleotides of the target sequence, respectively. Also described herein are DNAs encoding the shortened Cas9 guide RNAs that have been described previously in the literature (Jinek et al., Science. 337(6096):816-21 (2012) and Jinek et al., Elife. 2:e00471 (2013)).

The guide RNAs can include X_(N) which can be any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9.

In some embodiments, the guide RNA includes one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription.

Modified RNA oligonucleotides such as locked nucleic acids (LNAs) have been demonstrated to increase the specificity of RNA-DNA hybridization by locking the modified oligonucleotides in a more favorable (stable) conformation. For example, 2′-O-methyl RNA is a modified base where there is an additional covalent linkage between the 2′ oxygen and 4′ carbon which when incorporated into oligonucleotides can improve overall thermal stability and selectivity (formula I).

Thus in some embodiments, the tru-gRNAs disclosed herein may comprise one or more modified RNA oligonucleotides. For example, the truncated guide RNAs molecules described herein can have one, some or all of the 17-18 or 17-19 nts 5′ region of the guide RNA complementary to the target sequence are modified, e.g., locked (2′-O-4′-C methylene bridge), 5′-methylcytidine, 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), e.g., a synthetic ribonucleic acid.

In other embodiments, one, some or all of the nucleotides of the tru-gRNA sequence may be modified, e.g., locked (2′-O-4′-C methylene bridge), 5′-methylcytidine, 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), e.g., a synthetic ribonucleic acid.

In a cellular context, complexes of Cas9 with these synthetic gRNAs could be used to improve the genome-wide specificity of the CRISPR/Cas9 nuclease system.

Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA(X_(N)); (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG (X_(N)); (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU(X_(N)); (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAAC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more of the nucleotides are locked, e.g., one or more of the nucleotides within the sequence X₁₇₋₁₈ or X₁₇₋₁₉, one or more of the nucleotides within the sequence X_(N), or one or more of the nucleotides within any sequence of the tru-gRNA. X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. Although some of the examples described herein utilize a single gRNA, the methods can also be used with dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems). In this case, a single tracrRNA would be used in conjunction with multiple different crRNAs expressed using the present system, e.g., the following: (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404); (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU (SEQ ID NO:2408); and a tracrRNA sequence. In this case, the crRNA is used as the guide RNA in the methods and molecules described herein, and the tracrRNA can be expressed from the same or a different DNA molecule. In some embodiments, the methods include contacting the cell with a tracrRNA comprising or consisting of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof (an active portion is one that retains the ability to form complexes with Cas9 or dCas9). In some embodiments, the tracrRNA molecule may be truncated from its 3′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In another embodiment, the tracrRNA molecule may be truncated from its 5′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the tracrRNA molecule may be truncated from both the 5′ and 3′ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts on the 5′ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3′ end. Exemplary tracrRNA sequences in addition to SEQ ID NO:8 include the following: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof; AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof; CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof.

In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) is used as a crRNA, the following tracrRNA is used:

GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof. In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404) is used as a crRNA, the following tracrRNA is used: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof. In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) is used as a crRNA, the following tracrRNA is used: AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406) or an active portion thereof.

In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more modified (e.g., locked) nucleotides or deoxyribonucleotides.

In some embodiments, the single guide RNAs and/or crRNAs and/or tracrRNAs can include one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end.

Existing Cas9-based RGNs use gRNA-DNA heteroduplex formation to guide targeting to genomic sites of interest. However, RNA-DNA heteroduplexes can form a more promiscuous range of structures than their DNA-DNA counterparts. In effect, DNA-DNA duplexes are more sensitive to mismatches, suggesting that a DNA-guided nuclease may not bind as readily to off-target sequences, making them comparatively more specific than RNA-guided nucleases. Thus, the truncated guide RNAs described herein can be hybrids, i.e., wherein one or more deoxyribonucleotides, e.g., a short DNA oligonucleotide, replaces all or part of the gRNA, e.g., all or part of the complementarity region of a gRNA. This DNA-based molecule could replace either all or part of the gRNA in a single gRNA system or alternatively might replace all of part of the crRNA in a dual crRNA/tracrRNA system. Such a system that incorporates DNA into the complementarity region should more reliably target the intended genomic DNA sequences due to the general intolerance of DNA-DNA duplexes to mismatching compared to RNA-DNA duplexes. Methods for making such duplexes are known in the art, See, e.g., Barker et al., BMC Genomics. 2005 Apr. 22; 6:57; and Sugimoto et al., Biochemistry. 2000 Sep. 19; 39(37):11270-81.

Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:

(SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAAC AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAA GUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more of the nucleotides are deoxyribonucleotides, e.g., one or more of the nucleotides within the sequence X₁₇₋₁₈ or X₁₇₋₁₉, one or more of the nucleotides within the sequence X_(N), or one or more of the nucleotides within any sequence of the tru-gRNA. X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription.

In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more deoxyribonucleotides.

In some embodiments, the single guide RNAs or crRNAs or tracrRNAs includes one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end.

In some embodiments, the gRNA is targeted to a site that is at least three or more mismatches different from any sequence in the rest of the genome in order to minimize off-target effects.

The methods described can include expressing in a cell, or contacting the cell with, a shortened Cas9 gRNA (tru-gRNA) as described herein (optionally a modified or DNA/RNA hybrid tru-gRNA), plus a nuclease that can be guided by the shortened Cas9 gRNAs, e.g., a Cas9 nuclease, e.g., as described in Mali et al., a Cas9 nickase as described in Jinek et al., 2012; or a dCas9-heterofunctional domain fusion (dCas9-HFD).

Cas9

A number of bacteria express Cas9 protein variants. The Cas9 from Streptococcus pyogenes is presently the most commonly used; some of the other Cas9 proteins have high levels of sequence identity with the S. pyogenes Cas9 and use the same guide RNAs. Others are more diverse, use different gRNAs, and recognize different PAM sequences as well (the 2-5 nucleotide sequence specified by the protein which is adjacent to the sequence specified by the RNA). Chylinski et al. classified Cas9 proteins from a large group of bacteria (RNA Biology 10:5, 1-12; 2013), and a large number of Cas9 proteins are listed in supplementary FIG. 1 and supplementary table 1 thereof, which are incorporated by reference herein. Additional Cas9 proteins are described in Esvelt et al., Nat Methods. 2013 November; 10(11):1116-21 and Fonfara et al., “Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.” Nucleic Acids Res. 2013 Nov. 22. [Epub ahead of print] doi:10.1093/nar/gkt1074.

Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them. Such species include those set forth in the following table, which was created based on supplementary FIG. 1 of Chylinski et al., 2013.

Alternative Cas9 proteins GenBank Acc No. Bacterium 303229466 Veillonella atypica ACS-134-V-Col7a 34762592 Fusobacterium nucleatum subsp. vincentii 374307738 Filifactor alocis ATCC 35896 320528778 Solobacterium moorei F0204 291520705 Coprococcus catus GD-7 42525843 Treponema denticola ATCC 35405 304438954 Peptoniphilus duerdenii ATCC BAA-1640 224543312 Catenibacterium mitsuokai DSM 15897 24379809 Streptococcus mutans UA159 15675041 Streptococcus pyogenes SF370 16801805 Listeria innocua Clip11262 116628213 Streptococcus thermophilus LMD-9 323463801 Staphylococcus pseudintermedius ED99 352684361 Acidaminococcus intestini RyC-MR95 302336020 Olsenella uli DSM 7084 366983953 Oenococcus kitaharae DSM 17330 310286728 Bifidobacterium bifidum S17 258509199 Lactobacillus rhamnosus GG 300361537 Lactobacillus gasseri JV-V03 169823755 Finegoldia magna ATCC 29328 47458868 Mycoplasma mobile 163K 284931710 Mycoplasma gallisepticum str. F 363542550 Mycoplasma ovipneumoniae SC01 384393286 Mycoplasma canis PG 14 71894592 Mycoplasma synoviae 53 238924075 Eubacterium rectale ATCC 33656 116627542 Streptococcus thermophilus LMD-9 315149830 Enterococcus faecalis TX0012 315659848 Staphylococcus lugdunensis M23590 160915782 Eubacterium dolichum DSM 3991 336393381 Lactobacillus coryniformis subsp. torquens 310780384 Ilyobacter polytropus DSM 2926 325677756 Ruminococcus albus 8 187736489 Akkermansia muciniphila ATCC BAA-835 117929158 Acidothermus cellulolyticus 11B 189440764 Bifidobacterium longum DJO10A 283456135 Bifidobacterium dentium Bd1 38232678 Corynebacterium diphtheriae NCTC 13129 187250660 Elusimicrobium minutum Pei191 319957206 Nitratifractor salsuginis DSM 16511 325972003 Sphaerochaeta globus str. Buddy 261414553 Fibrobacter succinogenes subsp. succinogenes 60683389 Bacteroides fragilis NCTC 9343 256819408 Capnocytophaga ochracea DSM 7271 90425961 Rhodopseudomonas palustris BisB18 373501184 Prevotella micans F0438 294674019 Prevotella ruminicola 23 365959402 Flavobacterium columnare ATCC 49512 312879015 Aminomonas paucivorans DSM 12260 83591793 Rhodospirillum rubrum ATCC 11170 294086111 Candidatus Puniceispirillum marinum IMCC1322 121608211 Verminephrobacter eiseniae EF01-2 344171927 Ralstonia syzygii R24 159042956 Dinoroseobacter shibae DFL 12 288957741 Azospirillum sp- B510 92109262 Nitrobacter hamburgensis X14 148255343 Bradyrhizobium sp- BTAi1 34557790 Wolinella succinogenes DSM 1740 218563121 Campylobacter jejuni subsp. jejuni 291276265 Helicobacter mustelae 12198 229113166 Bacillus cereus Rock1-15 222109285 Acidovorax ebreus TPSY 189485225 uncultured Termite group 1 182624245 Clostridium perfringens D str. 220930482 Clostridium cellulolyticum H10 154250555 Parvibaculum lavamentivorans DS-1 257413184 Roseburia intestinalis L1-82 218767588 Neisseria meningitidis Z2491 15602992 Pasteurella multocida subsp. multocida 319941583 Sutterella wadsworthensis 3 1 254447899 gamma proteobacterium HTCC5015 54296138 Legionella pneumophila str. Paris 331001027 Parasutterella excrementihominis YIT 11859 34557932 Wolinella succinogenes DSM 1740 118497352 Francisella novicida U112 The constructs and methods described herein can include the use of any of those Cas9 proteins, and their corresponding guide RNAs or other guide RNAs that are compatible. The Cas9 from Streptococcus thermophilus LMD-9 CRISPR1 system has also been shown to function in human cells in Cong et al (Science 339, 819 (2013)). Cas9 orthologs from N. meningitides are described in Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9 and Esvelt et al., Nat Methods. 2013 November; 10(11):1116-21. Additionally, Jinek et al. showed in vitro that Cas9 orthologs from S. thermophilus and L. innocua, (but not from N. meningitidis or C. jejuni, which likely use a different guide RNA), can be guided by a dual S. pyogenes gRNA to cleave target plasmid DNA, albeit with slightly decreased efficiency.

In some embodiments, the present system utilizes the Cas9 protein from S. pyogenes, either as encoded in bacteria or codon-optimized for expression in mammalian cells, containing mutations at D10, E762, H983, or D986 and H840 or N863, e.g., D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive; substitutions at these positions could be alanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)) or they could be other residues, e.g., glutamine, asparagine, tyrosine, serine, or aspartate, e.g., E762Q, H983N, H983Y, D986N, N863D, N863S, or N863H (FIG. 1C). The sequence of the catalytically inactive S. pyogenes Cas9 that can be used in the methods and compositions described herein is as follows; the exemplary mutations of Dl OA and H840A are in bold and underlined.

        10         20         30         40 MDKKYSIGL A  IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR         50         60         70         80 HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC         90        100        110        120 YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG        130        140        150        160 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH        170        180        190        200 MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP        210        220        230        240 INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN        250        260        270        280 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA        290        300        310        320 QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS        330        340        350        360 MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA        370        380        390        400 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR        410        420        430        440 KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI        450        460        470        480 EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE        490        500        510        520 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV        530        540        550        560 YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT        570        580        590        600 VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI        610        620        630        640 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA        650        660        670        680 HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL        690        700        710        720 DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL        730        740        750        760 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV        770        780        790        800 IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP        810        820        830        840 VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVD A        850        860        870        880 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK        890        900        910        920 NYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ        930        940        950        960 LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS        970        980        990       1000 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK       1010       1020       1030       1040 YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS       1050       1060       1070       1080 NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF       1090       1100       1110       1120 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI       1130       1140       1150       1160 ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV       1170       1180       1190       1200 KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK       1210       1220       1230       1240 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS       1250       1260       1270       1280 HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV       1290       1300       1310       1320 ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA       1330       1340       1350       1360 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQ ID NO: 33)

In some embodiments, the Cas9 nuclease used herein is at least about 50% identical to the sequence of S. pyogenes Cas9, i.e., at least 50% identical to SEQ ID NO:33. In some embodiments, the nucleotide sequences are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:33. In some embodiments, any differences from SEQ ID NO:33 are in non-conserved regions, as identified by sequence alignment of sequences set forth in Chylinski et al., RNA Biology 10:5, 1-12; 2013 (e.g., in supplementary FIG. 1 and supplementary table 1 thereof); Esvelt et al., Nat Methods. 2013 November; 10(11):1116-21 and Fonfara et al., Nucl. Acids Res. (2014) 42 (4): 2577-2590. [Epub ahead of print 2013 Nov. 22] doi:10.1093/nar/gkt1074.

To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 50% (in some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 100% of the length of the reference sequence is aligned). The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For purposes of the present application, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Cas9-HFD

Cas9-HFD are described in a U.S. Provisional Patent Application Ser. No. 61/799,647, Filed on Mar. 15, 2013, U.S. Ser. No. 61/838,148, filed on Jun. 21, 2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.

The Cas9-HFD are created by fusing a heterologous functional domain (e.g., a transcriptional activation domain, e.g., from VP64 or NF-κB p65), to the N-terminus or C-terminus of a catalytically inactive Cas9 protein (dCas9). In the present case, as noted above, the dCas9 can be from any species but is preferably from S. pyogenes, In some embodiments, the Cas9 contains mutations in the D10 and H840 residues, e.g., D10N/D10A and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive, e.g., as shown in SEQ ID NO:33 above.

The transcriptional activation domains can be fused on the N or C terminus of the Cas9. In addition, although the present description exemplifies transcriptional activation domains, other heterologous functional domains (e.g., transcriptional repressors (e.g., KRAB, ERD, SID, and others, e.g., amino acids 473-530 of the ets2 repressor factor (ERF) repressor domain (ERD), amino acids 1-97 of the KRAB domain of KOX1, or amino acids 1-36 of the Mad mSIN3 interaction domain (SID); see Beerli et al., PNAS USA 95:14628-14633 (1998)) or silencers such as Heterochromatin Protein 1 (HP1, also known as swi6), e.g., HP1α or HP1β; proteins or peptides that could recruit long non-coding RNAs (lncRNAs) fused to a fixed RNA binding sequence such as those bound by the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein; enzymes that modify the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins); or enzymes that modify histone subunits (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), histone methyltransferases (e.g., for methylation of lysine or arginine residues) or histone demethylases (e.g., for demethylation of lysine or arginine residues)) as are known in the art can also be used. A number of sequences for such domains are known in the art, e.g., a domain that catalyzes hydroxylation of methylated cytosines in DNA. Exemplary proteins include the Ten-Eleven-Translocation (TET) 1-3 family, enzymes that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA.

Sequences for human TET1-3 are known in the art and are shown in the following table:

GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP_085128.2 NM_030625.2 TET2* NP_001120680.1 (var 1) NM_001127208.2 NP_060098.3 (var 2) NM_017628.4 TET3 NP_659430.1 NM_144993.1 *Variant (1) represents the longer transcript and encodes the longer isoform (a). Variant (2) differs in the 5′ UTR and in the 3′ UTR and coding sequence compared to variant 1. The resulting isoform (b) is shorter and has a distinct C-terminus compared to isoform a.

In some embodiments, all or part of the full-length sequence of the catalytic domain can be included, e.g., a catalytic module comprising the cysteine-rich extension and the 2OGFeDO domain encoded by 7 highly conserved exons, e.g., the Tet1 catalytic domain comprising amino acids 1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprising amino acids 966-1678. See, e.g., FIG. 1 of Iyer et al., Cell Cycle. 2009 Jun. 1; 8(11):1698-710. Epub 2009 Jun. 27, for an alignment illustrating the key catalytic residues in all three Tet proteins, and the supplementary materials thereof (available at ftp site ftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html) for full length sequences (see, e.g., seq 2c); in some embodiments, the sequence includes amino acids 1418-2136 of Tet1 or the corresponding region in Tet2/3.

Other catalytic modules can be from the proteins identified in Iyer et al., 2009.

In some embodiments, the heterologous functional domain is a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the dCas9 gRNA targeting sequences. For example, a dCas9 fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (lncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibens et al., Biol. Cell 100:125-138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the protein can be targeted to the dCas9 binding site using the methods and compositions described herein. In some embodiments, the Csy4 is catalytically inactive.

In some embodiments, the fusion proteins include a linker between the dCas9 and the heterologous functional domains. Linkers that can be used in these fusion proteins (or between fusion proteins in a concatenated structure) can include any sequence that does not interfere with the function of the fusion proteins. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.e., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). In some embodiments, the linker comprises one or more units consisting of GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35) unit. Other linker sequences can also be used.

Expression Systems

In order to use the guide RNAs described, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the guide RNA can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the guide RNA for production of the guide RNA. The nucleic acid encoding the guide RNA can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a guide RNA is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the guide RNA is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the guide RNA. In addition, a preferred promoter for administration of the guide RNA can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the gRNA, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the gRNA, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include PMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

The vectors for expressing the guide RNAs can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of gRNAs in mammalian cells following plasmid transfection. Alternatively, a T7 promoter may be used, e.g., for in vitro transcription, and the RNA can be transcribed in vitro and purified. Vectors suitable for the expression of short RNAs, e.g., siRNAs, shRNAs, or other small RNAs, can be used.

Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the gRNA encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the gRNA.

The present invention includes the vectors and cells comprising the vectors.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 Assessing Specificity of RNA-Guided Endonucleases

CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. This example describes the use of a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs.

Materials and Methods

The following materials and methods were used in Example 1.

Construction of Guide RNAs

DNA oligonucleotides (Table A) harboring variable 20 nt sequences for Cas9 targeting were annealed to generate short double-strand DNA fragments with 4 bp overhangs compatible with ligation into BsmBI-digested plasmid pMLM3636. Cloning of these annealed oligonucleotides generates plasmids encoding a chimeric +103 single-chain guide RNA with 20 variable 5′ nucleotides under expression of a U6 promoter (Hwang et al., Nat Biotechnol 31, 227-229 (2013); Mali et al., Science 339, 823-826 (2013)). pMLM3636 and the expression plasmid pJDS246 (encoding a codon optimized version of Cas9) used in this study are both available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas).

TABLE A

Sequences of oligonucleotides used to generate expression plasmids encoding single gRNAs/variant single gRNAs targeted to sites in the EGFP reporter gene and single gRNAs targeted to six endogenous human gene targets. #, SEQ ID NO:.

EGFP Activity Assays

U2OS.EGFP cells harboring a single integrated copy of an EGFP-PEST fusion gene were cultured as previously described (Reyon et al., Nat Biotech 30, 460-465 (2012)). For transfections, 200,000 cells were Nucleofected with the indicated amounts of sgRNA expression plasmid and pJDS246 together with 30 ng of a Td-tomato-encoding plasmid using the SE Cell Line 4D-Nucleofector™ X Kit (Lonza) according to the manufacturer's protocol. Cells were analyzed 2 days post-transfection using a BD LSRII flow cytometer. Transfections for optimizing gRNA/Cas9 plasmid concentration were performed in triplicate and all other transfections were performed in duplicate.

PCR Amplification and Sequence Verification of Endogenous Human Genomic Sites

PCR reactions were performed using Phusion Hot Start II high-fidelity DNA polymerase (NEB) with PCR primers and conditions listed in Table B. Most loci amplified successfully using touchdown PCR (98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s]10 cycles, [98° C., 10 s; 62° C., 15 s; 72° C., 30 s]25 cycles). PCR for the remaining targets were performed with 35 cycles at a constant annealing temperature of 68° C. or 72° C. and 3% DMSO or 1M betaine, if necessary. PCR products were analyzed on a QIAXCEL capillary electrophoresis system to verify both size and purity. Validated products were treated with ExoSap-IT (Affymetrix) and sequenced by the Sanger method (MGH DNA Sequencing Core) to verify each target site.

TABLE B

Sequences and characteristics of genomic on- and off-target sites for six RGNs targeted to endogenous human genes and primers and PCR conditions used to amplify these sites.

Determination of RGN-Induced on- and Off-Target Mutation Frequencies in Human Cells

For U2OS.EGFP and K562 cells, 2×10⁵ cells were transfected with 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for negative controls), 750 ng of Cas9 expression plasmid, and 30 ng of td-Tomato expression plasmid using the 4D Nucleofector System according to the manufacturer's instructions (Lonza). For HEK293 cells, 1.65×10⁵ cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for the negative control), 375 ng of Cas9 expression plasmid, and 30 ng of a td-Tomato expression plasmid using Lipofectamine LTX reagent according to the manufacturer's instructions (Life Technologies). Genomic DNA was harvested from transfected U2OS.EGFP, HEK293, or K562 cells using the QIAamp DNA Blood Mini Kit (QIAGEN), according to the manufacturer's instructions. To generate enough genomic DNA to amplify the off-target candidate sites, DNA from three Nucleofections (for U2OS.EGFP cells), two Nucleofections (for K562 cells), or two Lipofectamine LTX transfections was pooled together before performing T7EI. This was done twice for each condition tested, thereby generating duplicate pools of genomic DNA representing a total of four or six individual transfections. PCR was then performed using these genomic DNAs as templates as described above and purified using Ampure XP beads (Agencourt) according to the manufacturer's instructions. T7EI assays were performed as previously described (Reyon et al., 2012, supra).

DNA Sequencing of NHEJ-Mediated Indel Mutations

Purified PCR products used for the T7EI assay were cloned into Zero Blunt TOPO vector (Life Technologies) and plasmid DNAs were isolated using an alkaline lysis miniprep method by the MGH DNA Automation Core. Plasmids were sequenced using an M13 forward primer (5′-GTAAAACGACGGCCAG-3′ (SEQ ID NO:1059) by the Sanger method (MGH DNA Sequencing Core).

Example 1a Single Nucleotide Mismatches

To begin to define the specificity determinants of RGNs in human cells, a large-scale test was performed to assess the effects of systematically mismatching various positions within multiple gRNA/target DNA interfaces. To do this, a quantitative human cell-based enhanced green fluorescent protein (EGFP) disruption assay previously described (see Methods above and Reyon et al., 2012, supra) that enables rapid quantitation of targeted nuclease activities (FIG. 2B) was used. In this assay, the activities of nucleases targeted to a single integrated EGFP reporter gene can be quantified by assessing loss of fluorescence signal in human U2OS.EGFP cells caused by inactivating frameshift insertion/deletion (indel) mutations introduced by error prone non-homologous end joining (NHEJ) repair of nuclease-induced double-stranded breaks (DSBs) (FIG. 2B). For the studies described here, three ˜100 nt single gRNAs targeted to different sequences within EGFP were used, as follows:

(SEQ ID NO: 9) EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO: 10) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO: 11) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG Each of these gRNAs can efficiently direct Cas9-mediated disruption of EGFP expression (see Example 1e and 2a, and FIGS. 3E (top) and 3F (top)).

In initial experiments, the effects of single nucleotide mismatches at 19 of 20 nucleotides in the complementary targeting region of three EGFP-targeted gRNAs were tested. To do this, variant gRNAs were generated for each of the three target sites harboring Watson-Crick transversion mismatches at positions 1 through 19 (numbered 1 to 20 in the 3′ to 5′ direction; see FIG. 1) and the abilities of these various gRNAs to direct Cas9-mediated EGFP disruption in human cells tested (variant gRNAs bearing a substitution at position 20 were not generated because this nucleotide is part of the U6 promoter sequence and therefore must remain a guanine to avoid affecting expression.)

For EGFP target site #2, single mismatches in positions 1-10 of the gRNA have dramatic effects on associated Cas9 activity (FIG. 2C, middle panel), consistent with previous studies that suggest mismatches at the 5′ end of gRNAs are better tolerated than those at the 3′ end (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al., Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)). However, with EGFP target sites #1 and #3, single mismatches at all but a few positions in the gRNA appear to be well tolerated, even within the 3′ end of the sequence. Furthermore, the specific positions that were sensitive to mismatch differed for these two targets (FIG. 2C, compare top and bottom panels)—for example, target site #1 was particularly sensitive to a mismatch at position 2 whereas target site #3 was most sensitive to mismatches at positions 1 and 8.

Example 1b Multiple Mismatches

To test the effects of more than one mismatch at the gRNA/DNA interface, a series of variant gRNAs bearing double Watson-Crick transversion mismatches in adjacent and separated positions were created and the abilities of these gRNAs to direct Cas9 nuclease activity were tested in human cells using the EGFP disruption assay. All three target sites generally showed greater sensitivity to double alterations in which one or both mismatches occur within the 3′ half of the gRNA targeting region. However, the magnitude of these effects exhibited site-specific variation, with target site #2 showing the greatest sensitivity to these double mismatches and target site #1 generally showing the least. To test the number of adjacent mismatches that can be tolerated, variant gRNAs were constructed bearing increasing numbers of mismatched positions ranging from positions 19 to 15 in the 5′ end of the gRNA targeting region (where single and double mismatches appeared to be better tolerated).

Testing of these increasingly mismatched gRNAs revealed that for all three target sites, the introduction of three or more adjacent mismatches results in significant loss of RGN activity. A sudden drop off in activity occurred for three different EGFP-targeted gRNAs as one makes progressive mismatches starting from position 19 in the 5′ end and adding more mismatches moving toward the 3′ end. Specifically, gRNAs containing mismatches at positions 19 and 19+18 show essentially full activity whereas those with mismatches at positions 19+18+17, 19+18+17+16, and 19+18+17+16+15 show essentially no difference relative to a negative control (FIG. 2F). (Note that we did not mismatch position 20 in these variant gRNAs because this position needs to remain as a G because it is part of the U6 promoter that drives expression of the gRNA.)

Additional proof of that shortening gRNA complementarity might lead to RGNs with greater specificities was obtained in the following experiment: for four different EGFP-targeted gRNAs (FIG. 2H), introduction of a double mismatch at positions 18 and 19 did not significantly impact activity. However, introduction of another double mismatch at positions 10 and 11 then into these gRNAs results in near complete loss of activity. Interestingly introduction of only the 10/11 double mismatches does not generally have as great an impact on activity.

Taken together, these results in human cells confirm that the activities of RGNs can be more sensitive to mismatches in the 3′ half of the gRNA targeting sequence. However, the data also clearly reveal that the specificity of RGNs is complex and target site-dependent, with single and double mismatches often well tolerated even when one or more mismatches occur in the 3′ half of the gRNA targeting region. Furthermore, these data also suggest that not all mismatches in the 5′ half of the gRNA/DNA interface are necessarily well tolerated.

In addition, these results strongly suggest that gRNAs bearing shorter regions of complementarity (specifically ˜17 nts) will be more specific in their activities. We note that 17 nts of specificity combined with the 2 nts of specificity conferred by the PAM sequence results in specification of a 19 bp sequence, one of sufficient length to be unique in large complex genomes such as those found in human cells.

Example 1c Off-Target Mutations

To determine whether off-target mutations for RGNs targeted to endogenous human genes could be identified, six single gRNAs that target three different sites in the VEGFA gene, one in the EMX1 gene, one in the RNF2 gene, and one in the FANCF gene were used (Table 1 and Table A). These six gRNAs efficiently directed Cas9-mediated indels at their respective endogenous loci in human U2OS.EGFP cells as detected by T7 Endonuclease I (T7EI) assay (Methods above and Table 1). For each of these six RGNs, we then examined dozens of potential off-target sites (ranging in number from 46 to as many as 64) for evidence of nuclease-induced NHEJ-mediated indel mutations in U2OS.EGFP cells. The loci assessed included all genomic sites that differ by one or two nucleotides as well as subsets of genomic sites that differ by three to six nucleotides and with a bias toward those that had one or more of these mismatches in the 5′ half of the gRNA targeting sequence (Table B). Using the T7EI assay, four off-target sites (out of 53 candidate sites examined) for VEGFA site 1, twelve (out of 46 examined) for VEGFA site 2, seven (out of 64 examined) for VEGFA site 3 and one (out of 46 examined) for the EMX1 site (Table 1 and Table B) were readily identified. No off-target mutations were detected among the 43 and 50 potential sites examined for the RNF2 or FANCF genes, respectively (Table B). The rates of mutation at verified off-target sites were very high, ranging from 5.6% to 125% (mean of 40%) of the rate observed at the intended target site (Table 1). These bona fide off-targets included sequences with mismatches in the 3′ end of the target site and with as many as a total of five mismatches, with most off-target sites occurring within protein coding genes (Table 1). DNA sequencing of a subset of off-target sites provided additional molecular confirmation that indel mutations occur at the expected RGN cleavage site (FIGS. 8A-C).

TABLE 1 On- and off-target mutations induced by RGNs designed to endogenous human genes SEQ Site ID Indel Mutation Frequency (%) ± SEM Target name Sequence NO: U20S.EGFP HEK293 K562 Gene Target 1 T1 GGGTGGGGGGAGTTTGCTCCTGG 1059. 26.0 ± 2.9 10.5 ± 0.07 3.33 ± 0.42 VEGFA (VEGFA Site 1) OT1-3 GG A TGG A GGGAGTTTGCTCCTGG 1060. 25.7 ± 9.1 18.9 ± 0.77 2.93 ± 0.04 IGDCC3 OT1-4 GGG A GGG T GGAGTTTGCTCCTGG 1061.  9.2 ± 0.8 8.32 ± 0.51 N.D. LOC116437 OT1-6 C GG G GG A GGGAGTTTGCTCCTGG 1062.  5.3 ± 0.2 3.67 ± 0.09 N.D. CACNA2D OT1-11 GGG GA GGGG A AGTTTGCTCCTGG 1063. 17.1 ± 4.7 8.54 ± 0.16 N.D. Target 2 T2 GACCCCCTCCACCCCGCCTCCGG 1064. 50.2 ± 4.9 38.6 ± 1.92 15.0 ± 0.25 VEGFA (VEGFA Site 2) OT2-1 GACCCCC C CCACCCCGCCCCCGG 1065. 14.4 ± 3.4 33.6 ± 1.17 4.10 ± 0.05 FMN1 OT2-2 G GG CCCCTCCACCCCGCCTCTGG 1066. 20.0 ± 6.2 15.6 ± 0.30 3.00 ± 0.06 PAX6 OT2-6 CTA CCCCTCCACCCCGCCTCCGG 1067.  8.2 ± 1.4 15.0 ± 0.64 5.24 ± 0.22 PAPD7 OT2-9 G C CCCC AC CCACCCCGCCTCTGG 1068. 50.7 ± 5.6 30.7 ± 1.44 7.05 ± 0.48 LAMAS OT2-15 T ACCCCC CA CACCCCGCCTCTGG 1069.  9.7 ± 4.5 6.97 ± 0.10 1.34 ± 0.15 SPNS3 OT2-17 ACA CCCC C CCACCCCGCCTCAGG 1070. 14.0 ± 2.8 12.3 ± 0.45 1.80 ± 0.03 012-19 ATT CCCC C CCACCCCGCCTCAGG 1071. 17.0 ± 3.3 19.4 ± 1.35 N.D. HDLBP OT2-20 CC CC A CC C CCACCCCGCCTCAGG 1072.  6.1 ± 1.3 N.D. N.D. ABLIM1 OT2-23 CG CCC T C C CCACCCCGCCTCCGG 1073. 44.4 ± 6.7 28.7 ± 1.15 4.18 ± 0.37 CALY OT2-24 CT CCCC AC CCACCCCGCCTCAGG 1074. 62.8 ± 5.0 29.8 ± 1.08 21.1 ± 1.68 OT2-29 TG CCCC TC CCACCCCGCCTCTGG 1075. 13.8 ± 5.2 N.D. N.D. ACLY OT2-34 AGG CCCC CA CACCCCGCCTCAGG 1076.  2.8 ± 1.5 N.D. N.D. Target 3 T3 GGTGAGTGAGTGTGTGCGTGTGG 1077. 49.4 ± 3.8 35.7 ± 1.26 27.9 ± 0.52 VEGFA (VEGFA Site 3) OT3-1 GGTGAGTGAGTGTGTG T GTGAGG 1078.  7.4 ± 3.4 8.97 ± 0.80 N.D. (abParts) OT3-2 A GTGAGTGAGTGTGTG T GTGGGG 1079. 24.3 ± 9.2 23.9 ± 0.08  8.9 ± 0.16 MAX OT3-4 G C TGAGTGAGTGT A TGCGTGTGG 1080.  20.9 ± 11.8 11.2 ± 0.23 N.D. OT3-9 GGTGAGTGAGTG C GTGCG G GTGG 1081.  3.2 ± 0.3 2.34 ± 0.21 N.D. TPCN2 OT3-17 G T TGAGTGA A TGTGTGCGTGAGG 1082.  2.9 ± 0.2 1.27 ± 0.02 N.D. SLIT1 OT3-18 T GTG G GTGAGTGTGTGCGTGAGG 1083. 13.4 ± 4.2 12.1 ± 0.24 2.42 ± 0.07 COMDA OT3-20 A G A GAGTGAGTGTGTGC A TGAGG 1084. 16.7 ± 3.5 7.64 ± 0.05 1.18 ± 0.01 Target 4 T4 GAGTCCGAGCAGAAGAAGAAGGG 1085. 42.1 ± 0.4 26.0 ± 0.70 10.7 ± 0.50 EMX1 (EMX1) OT4-1 GAGT TA GAGCAGAAGAAGAAAGG 1086. 16.8 ± 0.2 8.43 ± 1.32 2.54 ± 0.02 HCN1 Target 5 (RNF2) T5 GTCATCTTAGTCATTACCTGTGG 1087. 26.6 ± 6.0 --- --- RNF2 Target 6 (FANCF) T6 GGAATCCCTTCTGCAGCACCAGG 1088. 33.2 ± 6.5 --- --- FANCF “OT” indicates off-target sites (with numbering of sites as in Table E). Mismatches from the on-target (within the 20 by region to which the gRNA hybridizes) are highlighted as bold, 5 underlined text. Mean indel mutation frequencies in U2OS.EGFP, HEK293, and K562 cells were determined as described in Methods. Genes in which sites were located (if any) are shown. All sites listed failed to show any evidence of modification in cells transfected with Cas9 expression plasmid and a control U6 promoter plasmid that did not express a functional gRNA. N.D. = none detected; --- = not tested.

Example 1d Off-Target Mutations in Other Cell Types

Having established that RGNs can induce off-target mutations with high frequencies in U2OS.EGFP cells, we next sought to determine whether these nucleases would also have these effects in other types of human cells. We had chosen U2OS.EGFP cells for our initial experiments because we previously used these cells to evaluate the activities of TALENs¹⁵ but human HEK293 and K562 cells have been more widely used to test the activities of targeted nucleases. Therefore, we also assessed the activities of the four RGNs targeted to VEGFA sites 1, 2, and 3 and the EMX1 site in HEK293 and K562 cells. We found that each of these four RGNs efficiently induced NHEJ-mediated indel mutations at their intended on-target site in these two additional human cell lines (as assessed by T7EI assay) (Table 1), albeit with somewhat lower mutation frequencies than those observed in U2OS.EGFP cells. Assessment of the 24 off-target sites for these four RGNs originally identified in U2OS.EGFP cells revealed that many were again mutated in HEK293 and K562 cells with frequencies similar to those at their corresponding on-target site (Table 1). As expected, DNA sequencing of a subset of these off-target sites from HEK293 cells provided additional molecular evidence that alterations are occurring at the expected genomic loci (FIGS. 9A-C). We do not know for certain why in HEK293 cells four and in K562 cells eleven of the off-target sites identified in U2OS.EGFP cells did not show detectable mutations. However, we note that many of these off-target sites also showed relatively lower mutation frequencies in U2OS.EGFP cells. Therefore, we speculate that mutation rates of these sites in HEK293 and K562 cells may be falling below the reliable detection limit of our T7EI assay (˜2-5%) because RGNs generally appear to have lower activities in HEK293 and K562 cells compared with U2OS.EGFP cells in our experiments. Taken together, our results in HEK293 and K562 cells provide evidence that the high-frequency off-target mutations we observe with RGNs will be a general phenomenon seen in multiple human cell types.

Example 1e Titration of gRNA- and Cas9-Expressing Plasmid Amounts Used for the EGFP Disruption Assay

Single gRNAs were generated for three different sequences (EGFP SITES 1-3, shown above) located upstream of EGFP nucleotide 502, a position at which the introduction of frameshift mutations via non-homologous end joining can robustly disrupt expression of EGFP (Maeder, M. L. et al., Mol Cell 31, 294-301 (2008); Reyon, D. et al., Nat Biotech 30, 460-465 (2012)).

For each of the three target sites, a range of gRNA-expressing plasmid amounts (12.5 to 250 ng) was initially transfected together with 750 ng of a plasmid expressing a codon-optimized version of the Cas9 nuclease into our U2OS.EGFP reporter cells bearing a single copy, constitutively expressed EGFP-PEST reporter gene. All three RGNs efficiently disrupted EGFP expression at the highest concentration of gRNA-encoding plasmid (250 ng) (FIG. 3E (top)). However, RGNs for target sites #1 and #3 exhibited equivalent levels of disruption when lower amounts of gRNA-expressing plasmid were transfected whereas RGN activity at target site #2 dropped immediately when the amount of gRNA-expressing plasmid transfected was decreased (FIG. 3E(top)).

The amount of Cas9-encoding plasmid (range from 50 ng to 750 ng) transfected into our U2OS.EGFP reporter cells was titrated and EGFP disruption assayed. As shown in FIG. 3F (top), target site #1 tolerated a three-fold decrease in the amount of Cas9-encoding plasmid transfected without substantial loss of EGFP disruption activity. However, the activities of RGNs targeting target sites #2 and #3 decreased immediately with a three-fold reduction in the amount of Cas9 plasmid transfected (FIG. 3F (top)). Based on these results, 25 ng/250 ng, 250 ng/750 ng, and 200 ng/750 ng of gRNA-/Cas9-expressing plasmids were used for EGFP target sites #1, #2, and #3, respectively, for the experiments described in Examples 1a-1d.

The reasons why some gRNA/Cas9 combinations work better than others in disrupting EGFP expression is not understood, nor is why some of these combinations are more or less sensitive to the amount of plasmids used for transfection. Although it is possible that the range of off-target sites present in the genome for these three gRNAs might influence each of their activities, no differences were seen in the numbers of genomic sites that differ by one to six bps for each of these particular target sites (Table C) that would account for the differential behavior of the three gRNAs.

TABLE C Numbers of off-target sites in the human genome for six RGNs targeted to endogenous human genes and three RGNs targeted to the EGFP reporter gene Number of mismatches to on-target site Target Site 0 1 2 3 4 5 6 Target 1 (VEGFA Site 1) 1 1 4 32 280 2175 13873 Target 2 (VEGFA Site 2) 1 0 2 35 443 3889 17398 Target 3 (VEGFA Site 3) 1 1 17 377 6028 13398 35517 Target 4 (EMX) 1 0 1 18 276 2309 15731 Target 5 (RNF2) 1 0 0 6 116 976 7443 Target 6 (FANCF) 1 0 1 18 271 1467 9551 EGFP Target Site #1 0 0 3 10 156 1365 9755 EGFP Target Site #2 0 0 0 11 96 974 7353 EGFP Target Site #3 0 0 1 14 165 1439 10361 Off-target sites for each of the six RGNs targeted to the VEGFA, RNF2, FANCF, and EMX1 genes and the three RGNs targeted to EGFP Target Sites #1, #2 and #3 were identified in human genome sequence build GRCh37. Mismatches were only allowed for the 20 nt region to which the gRNA anneals and not to the PAM sequence.

Example 2 Shortening gRNA Complementarity Length to Improve RGN Cleavage Specificity

It was hypothesized that off-target effects of RGNs might be minimized without compromising on-target activity simply by decreasing the length of the gRNA-DNA interface, an approach that at first might seem counterintuitive. Longer gRNAs can actually function less efficiently at the on-target site (see below and Hwang et al., 2013a; Ran et al., 2013). In contrast, as shown above in Example 1, gRNAs bearing multiple mismatches at their 5′ ends could still induce robust cleavage of their target sites (FIGS. 2A and 2C-2F), suggesting that these nucleotides might not be required for full on-target activity. Therefore, it was hypothesized that truncated gRNAs lacking these 5′ nucleotides might show activities comparable to full-length gRNAs (FIG. 2A). It was speculated that if the 5′ nucleotides of full-length gRNAs are not needed for on-target activity then their presence might also compensate for mismatches at other positions along the gRNA-target DNA interface. If this were true, it was hypothesized that gRNAs might have greater sensitivity to mismatches and thus might also induce substantially lower levels of Cas9-mediated off-target mutations (FIG. 2A).

Experimental Procedures

The following experimental procedures were used in Example 2.

Plasmid Construction

All gRNA expression plasmids were assembled by designing, synthesizing, annealing, and cloning pairs of oligonucleotides (IDT) harboring the complementarity region into plasmid pMLM3636 (available from Addgene) as described above (Example 1). The resulting gRNA expression vectors encode a ˜100 nt gRNA whose expression is driven by a human U6 promoter. The sequences of all oligonucleotides used to construct gRNA expression vectors are shown in Table D. The Cas9 D10A nickase expression plasmid (pJDS271) bearing a mutation in the RuvC endonuclease domain was generated by mutating plasmid pJDS246 using a QuikChange kit (Agilent Technologies) with the following primers: Cas9 D10A sense primer 5′-tggataaaaagtattctattggtttagccatcggcactaattccg-3′ (SEQ ID NO:1089); Cas9 D10A antisense primer 5′-cggaattagtgccgatggctaaaccaatagaatactttttatcca-3′ (SEQ ID NO:1090). All the targeted gRNA plasmids and the Cas9 nickase plasmids used in this study are available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas).

TABLE D Sequences of oligonucleotides used to construct gRNA expression plasmids

Human Cell-Based EGFP Disruption Assay

U2OS.EGFP cells harboring a single-copy, integrated EGFP-PEST gene reporter have been previously described (Reyon et al., 2012). These cells were maintained in Advanced DMEM (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies), penicillin/streptomycin and 400 μg/ml G418. To assay for disruption of EGFP expression, 2×10⁵ U2OS.EGFP cells were transfected in duplicate with gRNA expression plasmid or an empty U6 promoter plasmid as a negative control, Cas9 expression plasmid (pJDS246) (Example 1 and Fu et al., 2013), and 10 ng of td-Tomato expression plasmid (to control for transfection efficiency) using a LONZA 4D-Nucleofector™, with SE solution and DN100 program according to the manufacturer's instructions. We used 25 ng/250 ng, 250 ng/750 ng, 200 ng/750 ng, and 250 ng/750 ng of gRNA expression plasmid/Cas9 expression plasmid for experiments with EGFP site #1, #2, #3, and #4, respectively. Two days following transfection, cells were trypsinized and resuspended in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and analyzed on a BD LSRII flow cytometer. For each sample, transfections and flow cytometry measurements were performed in duplicate.

Transfection of Human Cells and Isolation of Genomic DNA

To assess the on-target and off-target indel mutations induced by RGNs targeted to endogenous human genes, plasmids were transfected into U2OS.EGFP or HEK293 cells using the following conditions: U2OS.EGFP cells were transfected using the same conditions as for the EGFP disruption assay described above. HEK293 cells were transfected by seeding them at a density of 1.65×10⁵ cells per well in 24 well plates in Advanced DMEM (Life Technologies) supplemented with 10% FBS and 2 mM GlutaMax (Life Technologies) at 37° C. in a CO₂ incubator. After 22-24 hours of incubation, cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 375 ng of Cas9 expression plasmid (pJDS246) (Example 1 and Fu et al., 2013), and 10 ng of a td-Tomato expression plasmid, using Lipofectamine LTX reagent according to the manufacturer's instructions (Life Technologies). Medium was changed 16 hours after transfection. For both types of cells, genomic DNA was harvested two days post-transfection using an Agencourt DNAdvance genomic DNA isolation kit (Beckman) according to the manufacturer's instructions. For each RGN sample to be assayed, 12 individual 4D transfection replicates were performed, genomic DNA was isolated from each of these 12 transfections, and then these samples were combined to create two “duplicate” pools each consisting of six pooled genomic DNA samples. Indel mutations were then assessed at on-target and off-target sites from these duplicate samples by T7EI assay, Sanger sequencing, and/or deep sequencing as described below.

To assess frequencies of precise alterations introduced by HDR with ssODN donor templates, 2×10⁵ U2OS.EGFP cells were transfected 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 750 ng Cas9 expression plasmid (pJDS246), 50 pmol of ssODN donor (or no ssODN for controls), and 10 ng of td-Tomato expression plasmid (as the transfection control). Genomic DNA was purified three days after transfection using Agencourt DNAdvance and assayed for the introduction of a BamHI site at the locus of interest as described below. All of these transfections were performed in duplicate.

For experiments involving Cas9 nickases, 2×10⁵ U2OS.EGFP cells were transfected with 125 ng of each gRNA expression plasmid (if using paired gRNAs) or 250 ng of gRNA expression plasmid (if using a single gRNA), 750 ng of Cas9-D10A nickase expression plasmid (pJDS271), 10 ng of td-Tomato plasmid, and (if performing HDR) 50 pmol of ssODN donor template (encoding the BamHI site). All transfections were performed in duplicate. Genomic DNA harvested two days after transfection (if assaying for indel mutations) or three days after transfection (if assaying for HDR/ssODN-mediated alterations) using the Agencourt DNAdvance genomic DNA isolation kit (Beckman).

T7EI Assays for Quantifying Frequencies of Indel Mutations

T7EI assays were performed as previously described (Example 1 and Fu et al., 2013). In brief, PCR reactions to amplify specific on-target or off-target sites were performed with Phusion high-fidelity DNA polymerase (New England Biolabs) using one of the two following programs: (1) Touchdown PCR program [(98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s)×10 cycles, (98° C., 10 s; 62° C., 15 s; 72° C., 30 s)×25 cycles] or (2) Constant Tm PCR program [(98° C., 10 s; 68° C. or 72° C., 15 s; 72° C., 30 s)×35 cycles], with 3% DMSO or 1 M betaine if necessary. All primers used for these amplifications are listed in Table E. Resulting PCR products ranged in size from 300 to 800 bps and were purified by Ampure XP beads (Agencourt) according to the manufacturer's instructions. 200 ng of purified PCR products were hybridized in 1×NEB buffer 2 in a total volume of 19 μl and denatured to form heteroduplexes using the following conditions: 95° C., 5 minutes; 95 to 85° C., −2° C./s; 85 to 25° C., −0.1° C./s; hold at 4° C. 1 μl of T7 Endonuclease I (New England Biolabs, 10 units/μ1) was added to the hybridized PCR products and incubated at 37° C. for 15 minutes. The T7EI reaction was stopped by adding 2 μl of 0.25 M EDTA solution and the reaction products were purified using AMPure XP beads (Agencourt) with elution in 20 μl 0.1×EB buffer (QIAgen). Reactions products were then analyzed on a QIAXCEL capillary electrophoresis system and the frequencies of indel mutations were calculated using the same formula as previously described (Reyon et al., 2012).

TABLE E Mismatches Expected in target Off-Target compared non- Sequences SEQ to on- Actual Target SEQ Reverse SEQ Watson- Watson- Publication (Expected) - ID target in U2OS.EGFP Forward PCR ID PCR ID PCR Crick Crick ID HS GRCh37 NO: site cells Primer NO: Primer NO: Conditions Transversions Transversions Transitions Target 1 GGGTGGGGGGAG 1269. 0 TCCAGATGGCACA 1270. AGGGAGCA 1271. DMSO TTTGCTCCTGG TTGTCAG GGAAAGTG AGGT OT1-1 GGGTGGGGGGAG 1272. 1 GGGGCCCACTCTT 1273. ACCCAGAC 1279. No 0 0 1 TTTGCCCCAGG CTTCCAT TCCTGGTG DMSO TGGC OT1-2 GCGTGGGGGGTG 1275. 2 GCTAAGCAGAGAT 1276. ACCACCCT 1277. DMSO 2 0 0 TTTGCTCCCGG GCCTATGCC TTCCCCCA GAAA OT1-3 GGATGGAGGGAG 1278. 2 ACCCCACAGCCAG 1279. GAATCACT 1280. DMSO 0 0 2 TTTGCTCCTGG GTTTTCA GCACCTGG CCATC OT1-4 GGGAGGGTGGAG 1281. 2 TGCGGCAACTTCA 1282. TAAAGGGC 1283. DMSO 1 1 0 TTTGCTCCTGG GACAACC GTGCTGGG AGAG OT1-5 GGGTGGGTGGAG 1284. 2 GCATGTCAGGATC 1285. TGCAGGGC 1286. DMSO 0 2 0 TTTGCTACTGG TGACCCC CATCTTGT GTGT OT1-6 CGGGGGAGGGAG 1287. 3 CCACCACATGTTC 1288. CTGGGTCT 1289. DMSO 1 1 1 TTTGCTCCTGG TGGGTGC GTTCCCTG TGGG OT1-7 GAGTGGGTGGAG 1290. 3 GGCTCTCCCTGCC 1291. GCAGGTCA 1292. DMSO 0 2 1 TTTGCTACAGG CTAGTTT AGTTGGAA CCCG OT1-8 GGGAGGGGAGAG 1293. 3 GGGGCTGAGAACA 1294. AGATTTGT 1295. DMSO 1 0 2 TTTGTTCCAGG CATGAGATGCA GCACTGCC TGCCT OT1-9 GGGAGGGGGCAG 1296. 3 CCCGACCTCCGCT 1297. GGACCTCT 1298. DMSO 2 1 0 GTTGCTCCAGG CCAAAGC GCACACCC TGGC OT1-10 GGGAGGGGGGAG 1299. 3 TGCAAGGTCGCAT 1300. CAGGAGGG 1301. DMSO 1 1 1 TGTGTTCCGGG AGTCCCA GGAAGTGT GTCC OT1-11 GGGGAGGGGAAG 1302. 3 GCCCATTCTTTTT 1303. GAGAGCAA 1304. DMSO 0 1 2 TTTGCTCCAGG GCAGTGGA GTTTGTTC CCCAGG OT1-12 GGGGGTGGGGAC 1305. 3 GCCCCCAGCCCCT 1306. GCTGCTGG 1307. DMSO 1 2 0 TTTGCTCCAGG CTGTTTC TAGGGGAG CTGG OT1-13 GGGTCGGGGGAG 1308. 3 CGGCTGCCTTCCC 1309. GGGTGACG 1310. 72C 1 2 0 TGGGCTCCAGG TGAGTCC CTTGCCAT Anneal, GAGC 3% DMSO OT1-14 GGGTGGCTGGAG 1311. 3 TGACCCTGGAGTA 1312. GCTGAGAC 1313. 72C 2 1 0 TTTGCTGCTGG CAAAATGTTCCCA AACCAGCC Anneal, CAGCT 3% DMSO OT1-15 GGGTGGGGGGTG 1314. 3 TGCCTCCACCCTT 1315. GCAGCCGA 1316. DMSO 1 0 2 CCTGCTCCAGG AGCCCCT TCCACACT GGGG OT1-16 GGTTGAGGGGAG 1317. 3 AACTCAGGACAAC 1318. CCCAGGAG 1319. DMSO 0 1 2 TCTGCTCCAGG ACTGCCTGT CAGGGTAC AATGC OT1-17 GTGTGGGTGGCG 1320. 3 TCCTCCTTGGAGA 1321. CCTTGGAA 1322. DMSO 0 3 0 TTTGCTCCAGG GGGGCCC GGGGCCTT GGTGG OT1-18 AGGTGGTGGGAG 1323. 4 CCGAGGGCATGGG 1324. GGCTGCTG 1325. DMSO 0 1 3 CTTGTTCCTGG CAATCCT CGAGTTGC CAAC OT1-19 AGTTTGGGGGAG 1326. 4 TGCTTTGCATGGG 1327. GGGTTGCT 1328. DMSO 0 2 2 TTTGCCCCAGG GTCTCAGACA TGCCCTCT GTGT OT1-20 ATGTGTGGGGAA 1329. 4 AGCTCCTTCTCAT 1330. CACAGAAG 1331. DMSO 0 2 2 TTTGCTCCAGG TTCTCTTCTGCTG GATGTGTG T CAGGTT OT1-21 CAGTGGGGGGAG 1332. 4 AGCAGACACAGGT 1333. GGTCAGGT 1339. DMSO 1 1 2 CTTTCTCCTGG GAATGCTGCT GTGCTGCT AGGCA OT1-22 GAGGGGGAGCAG 1335. 4 CCTGTGGGGCTCT 1336. ACTGCCTG 1337. No 1 1 2 TTTGCTCCAGG CAGGTGC CCAAAGTG DMSO GGTGT TD OT1-23 GGAGGAGGGGAG 1338. 4 AGCTGCACTGGGG 1339. TGCCGGGT 1340. DMSO 0 1 3 TCTGCTCCAGG AATGAGT AATAGCTG GCTT OT1-24 GGAGGGGGGGCT 1341. 4 CCAGCCTGGGCAA 1342. GGGGGCTT 1343. 72C 0 3 1 TTTGCTCCAGG CAAAGCG CCAGGTCA Anneal, CAGG 3% DMSO, 6% DMSO OT1-25 GGGCAAGGGGAG 1344. 4 TACCCCCACTGCC 1345. ACAGGTCC 1346. DMSO 0 1 3 GTTGCTCCTGG CCATTGC ATGCTTAG CAGAGGG OT1-26 GGGTGATTGAAG 1347. 4 GGGTGATTGAAGTT ACGGATTCACGAC 1348. CCGAGTCC 1349. DMSO 0 / 1 2 2 TTTGCTCCAGG TGCTCCAGG (SEQ GGAGGTGC GTGGCAGA ID NO: 2225) GAGC GGGTGATTGAAGTT TGCTGCAGG (SEQ ID NO: 2226) OT1-27 GGGTGTGGGGTC 1350. 4 TGTGGTTGAAGTA 1351. TGGCCCAA 1352. DMSO 3 1 0 ATTGCTCCAGG GGGGACAGGT TTGGAAGT GATTTCGT OT1-28 GGTGGGGGTGGG 1353. 4 TGGGATGGCAGAG 1354. GGCCCAAT 1355. DMSO 0 3 1 TTTGCTCCAGG TCATCAACGT CGGTAGAG GATGCA OT1-29 GTGGGGGTAGAG 1356. 4 ATGGGGCGCTCCA 1357. TGCACCCA 1358. DMSO 0 3 1 TTTGCTCCAGG GTCTGTG CACAGCCA GCAA OT1-30 TAGTGGAGGGAG 1359. 4 GGGGAGGGAGGAC 1360. AATTAGCT 1361. 72C 0 1 3 CTTGCTCCTGG CAGGGAA GGGCGCGG Anneal, TGGT 3% DMSO OT1-31 TGCTCGGGGGAG 1362. 4 ATCCCGTGCAGGA 1363. CAGGCGGC 1364. DMSO 3 1 0 TTTGCACCAGG AGTCGCC CCCTTGAG GAAT OT1-32 TGGAGAGGGGAG 1365. 4 CCCCAACCCTTTG 1366. TGAGGAGA 1367. DMSO 1 2 1 TTGGCTCCTGG CTCAGCG ACACCACA GGCAGA OT1-33 TGGTGTTGGGAG 1368. 4 ATCGACGAGGAGG 1369. CCCCTCAC 1370. DMSO 0 3 1 TCTGCTCCAGG GGGCCTT TCAAGCAG GCCC OT1-34 TTGGGGGGGCAG 1371. 4 TGCTCAAGGGGCC 1372. CAGGGGCA 1373. No 1 3 0 TTTGCTCCTGG TGTTCCA GTGGCAGG DMSO AGTC OT1-35 AAGTAAGGGAAG 1374. 5 TGCCTGGCACGCA 1375. GGGAAGGG 1376. DMSO 0 0 5 TTTGCTCCTGG GTAGGTG GGAACAGG TGCA OT1-36 AGAAGAGGGGAT 1377. 5 Not optimized 1 1 3 TTTGCTCCTGG OT1-37 ATCTGGGGTGAT 1378. 5 ACCTGGGCTTGCC 1379. GCTGCTCG 1380. DMSO 1 3 1 TTTGCTCCTGG ACTAGGG CAGTTAAG CACCA OT1-38 CTCTGCTGGGAG 1381. 5 GTGGCCGGGCTAC 1382. GGTTCCAC 1383. DMSO 3 2 0 TTTGCTCCTGG TGCTACC AAGCTGGG GGCA OT1-39 CTGGTGGGGGAG 1384. 5 Not optimized 1 3 1 CTTGCTCCAGG OT1-40 CTTTCGGGGGAG 1385. 5 GCAAGAGGCGGAG 1386. AGAGTCAT 1387. DMSO 2 3 0 TTTGCGCCGGG GAGACCC CCATTTCC TGGGGGC OT1-41 CTTTGGGGTTAG 1388. 5 GGGGTCAGTGGTG 1389. AGGGAATC 1390. 1M 1 4 0 TTTGCTCCTGG ATATCCCCCT CTTTTTCC betaine, ATTGCTTG TD TTT OT1-42 GCTCTGGGGTAG 1391. 5 AGAGAGGCCACGT 1392. GCCTCCCC 1393. DMSO 1 3 1 TTTGCTCCAGG GGAGGGT TCCTCCTT CCCA OT1-43 GTCTCTCGGGAG 1394. 5 GACAGTGCCTTGC 1395. TCTGACCG 1396. DMSO 3 2 0 TTTGCTCCTGG GATGCAC GTATGCCT GACG OT1-44 TCCTGAGGGCAG 1397. 5 TGTGTGAACGCAG 1398. TGGTCTAG 1399. DMSO 3 1 1 TTTGCTCCAGG CCTGGCT TACTTCCT CCAGCCTT OT1-45 TCTTTGGGAGAG 1400. 5 GGTTCTCCCTTGG 1401. CCCACTGC 1402. DMSO 1 3 1 TTTGCTCCAGG CTCCTGTGA TCCTAGCC CTGC OT1-46 ACAACTGGGGAG 1403. 6 TGAAGTCAACAAT 1404. AGCTTTGG 1405. DMSO 3 1 2 TTTGCTCCTGG CTAAGCTTCCACC TAGTTGGA T GTCTTTGA AGG OT1-47 ACAAGGTGGAAG 1406. 6 TGATTGGGCTGCA 1407. GCACAGCC 1408. DMSO 2 1 3 TTTGCTCCTGG GTTCATGTACA TGCCCTTG GAAG OT1-48 ACATAGAAGGAG 1409. 6 TCCATGGGCCCCT 1410. AGCGGCTT 1411. DMSO 1 0 5 TTTGCTCCAGG CTGAAAGA CTGCTTCT GCGA OT1-49 AGACCCAGGGAG 1412. 6 GCGGTTGGTGGGG 1413. GAGTTCCT 1414. DMSO 2 0 4 TTTGCTCCCGG TTGATGC CCTCCCGC CAGT OT1-50 AGACCCAGGGAG 1415. 6 AGGCAAGATTTTC 1416. GCTTTTGC 1417. DMSO 2 0 4 TTTGCTCCCGG CAGTGTGCAAGA CTGGGACT CCGC No OT1-51 CACGGAGGGGTG 1418. 6 GCTGCTGGTCGGG 1419. GCTCTGTC 1420. DMSO 3 1 2 TTTGCTCCTGG CTCTCTG CCACTTCC TD CCTGG OT1-52 CAGAGCTTGGAG 1421. 6 GCTGCGAGGCTTC 1422. CGCCCCTA 1423. DMSO 3 2 1 TTTGCTCCAGG CGTGAGA GAGCTAAG GGGGT OT1-53 CTATTGATGGAG 1424. 6 CCAGGAGCCTGAG 1425. AGGGCTAG 1426. DMSO 1 3 2 TTTGCTCCTGG AGCTGCC GACTGCAG TGAGC OT1-54 CTTTCTAGGGAG 1427. 6 CTGTGCTCAGCCT 1428. GCCTGGGG 1429. DMSO 2 3 1 TTTGCTCCTGG GGGTGCT CTGTGAGT AGTTT OT1-55 GCCATGCTGGAG 1430. 6 AGCTCGCGCCAGA 1431. ACTTGGCA 1432. 72C 4 2 0 TTTGCTCCAGG TCTGTGG GGCTGAGG Anneal, CAGG 3% DMSO 1433. 1434. 1435. Target 2 GACCCCCTCCAC 1436. 0 AGAGAAGTCGAGG 1437. CAGCAGAA 1438. DMSO CCCGCCTCCGG AAGAGAGAG AGTTCATG GTTTCG OT2-1 GACCCCCCCCAC 1439. 2 TGGACAGCTGCAG 1440. ACTGATCG 1441. DMSO 0 0 2 CCCGCCCCCGG TACTCCCTG ATGATGGC CTATGGGT OT2-2 GGGCCCCTCCAC 1442. 2 CAAGATGTGCACT 1443. GCAGCCTA 1444. DMSO 1 0 1 CCCGCCTCTGG TGGGCTA TTGTCTCC TGGT OT2-3 AACCCCATCCAC 1445. 3 GTCCAGTGCCTGA 1446. AGCATCAT 1447. DMSO 1 1 1 CCGGCCTCAGG CCCTGGC GCCTCCAG CTTCA OT2-4 CACCCCCTCAAC 1448. 3 GCTCCCGATCCTC 1449. GCAGCTCC 1450. DMSO 1 2 0 ACCGCCTCAGG TGCCACC CACCACCC TCAG OT2-5 CACCCCCTCCCC 1451. 3 GGGGACAGGCAGG 1452. GTGCGTGT 1453. DMSO 1 1 1 TCCGCCTCAGG CAAGGAG CCGTTCAC CCCT OT2-6 CTACCCCTCCAC 1454. 3 AAGGGGCTGCTGG 1455. CGTGATTC 1456. DMSO 2 1 0 CCCGCCTCCGG GTAGGAC GAGTTCCT GGCA OT2-7 GACCCGCCCCGC 1457. 3 GACCCTCAGGAAG 1458. CTGCGAGA 1459. 1M 1 0 2 CCCGCCTCTGG CTGGGAG TGCCCCAA betaine, ATCG TD OT2-8 GATCGACTCCAC 1460. 3 CCGCGGCGCTCTG 1461. TGCTGGGA 1462. DMSO 1 1 1 CCCGCCTCTGG CTAGA TTACAGGC GCGA OT2-9 GCCCCCACCCAC 1463. 3 CCAGGTGGTGTCA 1464. TGCCTGGC 1465. DMSO 0 2 1 CCCGCCTCTGG GCGGAGG CCTCTCTG AGTCT OT2-10 GCCCCGCTCCTC 1466. 3 CGACTCCACGGCG 1467. CAGCGCAG 1468. 1M 2 1 0 CCCGCCTCCGG TCTCAGG TCCAGCCC betaine, GATG TD OT2-11 GGCCCCCTCCAC 1469. 3 CTTCCCTCCCCCA 1470. GCTACAGG 1471. DMSO 1 1 1 CAGGCCTCAGG GCACCAC TTGCACAG TGAGAGGT OT2-12 GGCCCCCTCCTC 1472. 3 CCCCGGGGAGTCT 1473. CCCAGCCG 1479. 72C 1 0 2 CTCGCCTCTGG GTCCTGA TTCCAGGT Anneal, CTTCC 3% DMSO OT2-13 GGCGCCCTCCAC 1475. 3 GAAGCGCGAAAAC 1476. TCCAGGGT 1477. DMSO 1 0 2 CCTGCCTCGGG CCGGCTC CCTTCTCG GCCC OT2-14 GTCCTCCACCAC 1478. 3 AGGGTGGTCAGGG 1479. CATGGGGC 1480. DMSO 2 0 1 CCCGCCTCTGG AGGCCTT TCGGACCT CGTC OT2-15 TACCCCCCACAC 1481. 3 GGGAAGAGGCAGG 1482. TGCCAGGA 1483. 72C 0 2 1 CCCGCCTCTGG GCTGTCG AGGAAGCT Anneal, GGCC 3% DMSO OT2-16 AACCCATTCCAC 1484. 4 GAGTGACGATGAG 1485. CCCTTAGC 1486. 68C 0 1 3 CCTGCCTCAGG CCCCGGG TGCAGTCG Anneal, CCCC 3% DMSO OT2-17 ACACCCCCCCAC 1487. 4 CCCATGAGGGGTT 1488. TGAAGATG 1489. DMSO 0 2 2 CCCGCCTCAGG TGAGTGC GGCAGTTT GGGG OT2-18 AGCCCCCACCTC 1490. 4 CACCTGGGGCATC 1491. ACTGGGGT 1492. DMSO 2 0 2 CCCGCCTCTGG TGGGTGG TGGGGAGG GGAT OT2-19 ATTCCCCCCCAC 1493. 4 TCATGATCCCCAA 1494. CCATTTGT 1495. DMSO 1 0 3 CCCGCCTCAGG AAGGGCT GCTGATCT GTGGGT OT2-20 CCCCACCCCCAC 1496. 4 TGGTGCCCAGAAT 1497. AGGAAATG 1498. DMSO 1 2 1 CCCGCCTCAGG AGTGGCCA TGTTGTGC CAGGGC OT2-21 CCCCCCCACCAC 1499. 4 GCCTCAGACAACC 1500. GCCAAGTG 1501. No 2 1 1 CCCGCCCCGGG CTGCCCC TTACTCAT DMSO CAAGAAAG TD TGG OT2-22 CCCCCCCCCCCC 1502. 4 GCCGGGACAAGAC 1503. TCCCGAAC 1504. DMSO 1 2 1 CCCGCCTCAGG TGAGTTGGG TCCCGCAA AACG OT2-23 CGCCCTCCCCAC 1505. 4 TGCTGCAGGTGGT 1506. CTGGAACC 1507. No 1 0 3 CCCGCCTCCGG TCCGGAG GCATCCTC DMSO CGCA TD OT2-24 CTCCCCACCCAC 1508. 4 ACACTGGTCCAGG 1509. GGCTGTGC 1510. DMSO 2 1 1 CCCGCCTCAGG TCCCGTCT CTTCCGAT GGAA OT2-25 CTCTCCCCCCAC 1511. 4 CTCTCCCCCCACCC ATCGCGCCCAAAG 1512. AGGCTTCT 1513. DMSO 3 0 2 CCCGCCTCTGG CCCCTCTGG (SEQ CACAGGT GGAAAAGT ID NO: 2227) CCTCAATG CA OT2-26 GCCTCTCTGCAC 1514. 4 Not optimized 1 1 2 CCCGCCTCAGG OT2-27 GTCACTCCCCAC 1515. 4 CCCTCATGGTGGT 1516. AGCCACAC 1517. DMSO 1 1 2 CCCGCCTCTGG CTTACGGCA ATCTTTCT GGTAGGG OT2-28 TGCCCCCTCCCC 1518. 4 TGCGTCGCTCATG 1519. AGGGTGGG 1520. DMSO 0 3 1 CCAGCCTCTGG CTGGGAG GTGTACTG GCTCA OT2-29 TGCCCCTCCCAC 1521. 4 GAGCTGAGACGGC 1522. TGGCCTTG 1523. 1M 0 1 3 CCCGCCTCTGG ACCACTG AACTCTTG betaine, GGCT TD OT2-30 TTCCCCTTCCAC 1524. 4 Not optimized 1 2 1 CCAGCCTCTGG OT2-31 TTCTCCCTCCTC 1525. 4 AGTGAGAGTGGCA 1526. CAGTAGGT 1527. DMSO 2 1 1 CCCGCCTCGGG CGAACCA GGTCCCTT CCGC OT2-32 ACCCTCGCCCAC 1528. 5 Not optimized 1 1 3 CCCGCCTCAGG OT2-33 AGCCAACCCCAC 1529. 5 GGGAGAACCTTGT 1530. AAGCCGAA 1531. DMSO 0 2 3 CCCGCCTCTGG CCAGCCT AAGCTGGG CAAA OT2-34 AGGCCCCCACAC 1532. 5 CTTCCCAGTGTGG 1533. ACACAGTC 1534. DMSO 1 1 3 CCCGCCTCAGG CCCGTCC AGAGCTCC GCCG OT2-35 AGGCCCCCCCGC 1535. 5 Not optimized 1 0 4 CCCGCCTCAGG OT2-36 ATCTGCCACCAC 1536. 5 CTGAGAGGGGGAG 1537. TCGACTGG 1538. 68C 3 0 2 CCCGCCTCAGG GGGGAGG TCTTGTCC Anneal, TCCCA 3% DMSO OT2-37 CATCTTCCCCAC 1539. 5 CAGCCTGCTGCAT 1540. TGCAGCCA 1541. 1M 1 0 4 CCCGCCTCTGG CGGAAAA AGAGAAAA betaine, AGCCT TD OT2-38 CTTTCCCTCCAC 1542. 5 TCCCTCTGACCCG 1543. ACCCGACT 1544. DMSO 2 1 2 CCAGCCTCTGG GAACCCA TCCTCCCC ATTGC OT2-39 GTCGAGGTCCAC 1545. 5 TGGGGGTTGCGTG 1546. GCCAGGAG 1547. DMSO 4 1 0 CCCGCCTCAGG CTTGTCA GACACCAG GACC OT2-40 GTCGAGGTCCAC 1548. 5 ATCAGGTGCCAGG 1549. GGCCTGAG 1550. DMSO 4 1 0 CCCGCCTCAGG AGGACAC AGTGGAGA GTGG OT2-41 TCAGACCTCCAC 1551. 5 Not optimized 1 4 0 CCCGCCTCAGG OT2-42 TGCAACCTCCTC 1552. 5 TGAGCCACATGAA 1553. ACCTCTCC 1554. DMSO 1 3 1 CCCGCCTCGGG TCAAGGCCTCC AAGTCTCA GTAACTCT CT OT2-43 ACCAGTCTGCAC 1555. 6 GGTCCCTCTGTGC 1556. CTTTGGTG 1557. DMSO 2 2 2 CCCGCCTCTGG AGTGGAA GACCTGCA CAGC OT2-44 ACTACCCACCTC 1558. 6 GCGAGGCTGCTGA 1559. GCTGGGAC 1560. DMSO 2 2 2 CCCGCCTCAGG CTTCCCT TACAGACA TGTGCCA OT2-45 ATTTCCCCCCCC 1561. 6 ATTTCCTCCCCCCC ATTGCAGGCGTGT 1562. AAATCCTG 1563. DMSO 1 1 5 CCCGCCTCAGG C-CCTCAGG (SEQ CCAGGCA CATGGTGA ID NO: 2228) TGGGAGT OT2-46 CCACCATCCCAC 1564. 6 TGCTCTGCCATTT 1565. ACAGCCTC 1566. DMSO 1 3 2 CCCGCCTCTGG ATGTCCTATGAAC TTCTCCAT T GACTGAGC OT2-47 CCCAAGCCCCAC 1567. 6 TCCGCCCAAACAG 1568. GCGGTGGG 1569. DMSO 2 3 1 CCCGCCTCGGG GAGGCAG GAAGCCAT TGAG OT2-48 CCGCGCTTCCGC 1570. 6 GGGGGTCTGGCTC 1572. CCTGTCGG 1572. DMSO 3 1 2 CCCGCCTCTGG ACCTGGA GAGAGTGC CTGC OT2-49 CCTGCCATGCAC 1573. 6 TCCTGGTTCATTT 1574. ACTCCAGA 1575. DMSO 3 2 1 CCCGCCTCAGG GCTAGAACTCTGG TGCAACCA A GGGCT OT2-50 CTGCCTCCTCAC 1576. 6 CGTGTGGTGAGCC 1577. GCTTCACC 1578. DMSO 3 0 3 CCCGCCTCAGG TGAGTCT GTAGAGGC TGCT OT2-51 TCTTCTTTCCAC 1579. 6 AGGCCCTGATAAT 1580. TCAGTGAC 1581. DMSO 0 2 4 CCCGCCTCAGG TCATGCTACCAA AACCTTTT GTATTCGG CA OT2-52 TTGACCCCCCGC 1582. 6 Not optimized 2 2 2 CCCGCCTCAGG Target 3 GGTGAGTGAGTG 1583. 0 TCCAGATGGCACA 1564. AGGGAGCA 1565. DMSO TGTGCGTGTGG TTGTCAG GGAAAGTG AGGT OT3-1 GGTGAGTGAGTG 1586. 1 GCAGGCAAGCTGT 1587. CACCGACA 1588. DMSO 0 0 1 TGTGTGTGAGG CAAGGGT CACCCACT CACC OT3-2 AGTGAGTGAGTG 1589. 2 GAGGGGGAAGTCA 1590. TACCCGGG 1591. DMSO 0 0 2 TGTGTGTGGGG CCGACAA CCGTCTGT TAGA OT3-3 AGTGTGTGAGTG 1592. 2 GACACCCCACACA 1593. TGAATCCC 1599. DMSO 1 0 1 TGTGCGTGTGG CTCTCATGC TTCACCCC CAAG OT3-9 GCTGAGTGAGTG 1595. 2 TCCTTTGAGGTTC 1596. CCAATCCA 1597. DMSO 1 0 1 TATGCGTGTGG ATCCCCC GGATGATT CCGC OT3-5 GGTGAGTCAGTG 1598. 2 CAGGGCCAGGAAC 1599. GGGAGGTA 1600. DMSO 1 1 0 TGTGAGTGAGG ACAGGAA TGTGCGGG AGTG OT3-6 GGTGAGTGAGAG 1601. 2 TGCAGCCTGAGTG 1602. GCCCAGGT 1603. DMSO 1 0 1 TGTGTGTGTGG AGCAAGTGT GCTAAGCC CCTC OT3-7 GGTGAGTGAGTG 1604. 2 TACAGCCTGGGTG 1605. TGTGTCAT 1606. 1M 1 1 0 AGTGAGTGAGG ATGGAGC GGACTTTC betaine, CCATTGT TD OT3-8 GGTGAGTGAGTG 1607. 2 GGCAGGCATTAAA 1608. TCTCCCCC 1609. DMSO 1 1 0 AGTGAGTGAGG CTCATCAGGTCC AAGGTATC AGAGAGCT OT3-9 GGTGAGTGAGTG 1610. 2 GGGCCTCCCTGCT 1611. GCTGCCGT 1612. DMSO 0 1 1 CGTGCGGGTGG GGTTCTC CCGAACCC AAGA OT3-10 GGTGAGTGTGTG 1613. 2 ACAAACGCAGGTG 1614. ACTCCGAA 1615. DMSO 1 1 0 TGTGAGTGTGG GACCGAA AATGCCCC GCAGT OT3-11 GGTGAGTGTGTG 1616. 2 AGGGGAGGGGACA 1617. TTGAGAGG 1618. DMSO 1 0 1 TGTGCATGTGG TTGCCT GTTCAGTG GTTGC OT3-12 GGTGTGTGAGTG 1619. 2 CTAATGCTTACGG 1620. AGCCAACG 1621. DMSO 1 0 1 TGTGTGTGTGG CTGCGGG GCAGATGC AAAT OT3-13 GGTGTGTGTGTG 1622. 2 GAGCGAAGTTAAC 1623. CACACATG 1629. 68C, 2 0 0 TGTGCGTGCGG CCACCGC CACATGCC 3% CCTG DMSO OT3-14 GGTGTGTGTGTG 1625. 2 GCATGTGTCTAAC 1626. TCCCCCAT 1627. DMSO 2 0 0 TGTGCGTGTGG TGGAGACAATAGC ATCAACAC A ACACA OT3-15 GGTGTGTGTGTG 1628. 2 GCCCCTCCCGCCT 1629. TGGGCAAA 1630. DMSO 2 0 0 TGTGCGTGTGG TTTGTGT GGACATGA AACAGACA OT3-16 GGTGTGTGTGTG 1631. 2 GCCTCAGCTCTGC 1632. ACGAACAG 1633. DMSO 2 0 0 TGTGCGTGTGG TCTTAAGCCC ATCATTTT TCATGGCT TCC OT3-17 GTTGAGTGAATG 1634. 2 CTCCAGAGCCTGG 1635. CCCTCTCC 1636. DMSO 0 1 1 TGTGCGTGAGG CCTACCA GGAAGTGC CTTG OT3-18 TGTGGGTGAGTG 1637. 2 TCTGTCACCACAC 1638. GTTGCCTG 1639. DMSO 0 1 1 TGTGCGTGAGG AGTTACCACC GGGATGGG GTAT OT3-19 ACTGTGTGAGTG 1640. 3 GGGGACCCTCAAG 1691. GGGCATCA 1642. DMSO 2 0 1 TGTGCGTGAGG AGGCACT AAGGATGG GGAT OT3-20 AGAGAGTGAGTG 1643. 3 TGTGGAGGGTGGG 1694. ACAGTGAG 1645. DMSO 1 0 2 TGTGCATGAGG ACCTGGT GTGCGGTC TTTGGG OT3-21 AGCGAGTGGGTG 1646. 3 CGGGGTGGCAGTG 1697. GGTGCAGT 1698. DMSO 0 0 3 TGTGCGTGTGG ACGTCAA CCAAGAGC CCCC OT3-22 AGGGAGTGACTG 1649. 3 AGCTGAGGCAGAG 1650. GGGAGACA 1651. DMSO 1 1 1 TGTGCGTGTGG TCCCCGA GAGCAGCG CCTC OT3-23 AGTGAGTGAGTG 1652. 3 ACCACCAGACCCC 1653. AGGACGAC 1659. 72C 1 1 1 AGTGAGTGAGG ACCTCCA TTGTGCCC Anneal, CATTCA 3% DMSO OT3-24 CATGAGTGAGTG 1655. 3 GCGTCAGGACGCA 1656. TCCACCCA 1657. 72C 2 0 1 TGTGGGTGGGG GGTCAGA CCCACCCA Anneal, TCCT 3% DMSO OT3-25 CGTGAGTGTGTG 1658. 3 ACACTCTGGGCTA 1659. GCCCCCTC 1660. DMSO 2 0 1 TATGCGTGTGG GGTGCTGGA ACCACATG ATGCT OT3-26 GGACTGTGAGTG 1661. 3 GGGGCCATTCCTC 1662. TGGGGATC 1663. DMSO 3 0 0 TGTGCGTGAGG TGCTGCA CTTGCTCA TGGC OT3-27 GGTGTGTGCCTG 1664. 3 ACACACTGGCTCG 1665. CCTGCACG 1666. DMSO 2 1 0 TGTGCGTGTGG CATTCACCA AGGCCAGG TGTT OT3-28 GTTTCATGAGTG 1667. 3 TGGGCACGTAGTA 1668. CTCGCCGC 1669. DMSO 0 3 1 TGTGCGTGGGG AACTGCACCA CGTGACTG TAGG OT3-29 TGAGTGTGAGTG 1670. 3 TCAGCTGGTCCTG 1672. AGAGCACT 1672. DMSO 2 1 0 TGTGCGTGGGG GGCTTGG GGGTAGCA GTCAGT OT3-30 TGCCAGTGAGTG 1673. 3 AGACACAGCCAGG 1674. GGTGGGCG 1675. 68C, 1 1 1 TGTGCGTGTGG GCCTCAG TGTGTGTG 3% TACC DMSO OT3-31 TGGGTGTGAGTG 1676. 3 ACACTCTCACACA 1677. GAGAAGTC 1678. 72C 1 2 0 TGTGCGTGTGG CGCACCAA AGGGCTGG Anneal, CGCG 3% DMSO OT3-32 TGTATGTGAGTG 1679. 3 ACTGCCTGCATTT 1680. TGGTGAGG 1681. DMSO 1 1 1 TGTGCGTGTGG CCCCGGT GCTTCAGG GAGC OT3-33 TGTGAGAGAGAG 1682. 3 GCCAGGTTCATTG 1683. TCCTTCTA 1684. DMSO 2 1 0 TGTGCGTGTGG ACTGCCC CACATCGG CGGC OT3-34 TGTGCCTGAGTG 1685. 3 CGAGGGAGCCGAG 1686. CTGACCTG 1687. DMSO 1 2 0 TGTGCGTGTGG TTCGTAA GGGCTCTG GTAC OT3-35 TGTGTGTGTGTG 1688. 3 TCCTCGGGAAGTC 1689. GCACTGAG 1690. DMSO 2 1 0 TGTGCGTGTGG ATGGCTTCA CAACCAGG AGCAC OT3-36 AGCGTGTGAGTG 1691. 4 Not optimized 1 0 3 TATGCGTGGGG OT3-37 ATTGAGTGTGTG 1692. 4 TAAACCGTTGCCC 1693. GCTCCCCT 1694. DMSO 2 1 1 AGTGCGTGGGG CCGCCTC GCCAGGTG AACC OT3-38 CATGTGTGGGTG 1695. 4 CCTGCTGAGACTC 1696. CTGCGGAG 1697. DMSO 2 0 2 TGTGCGTGTGG CAGGTCC TGGCTGGC TATA OT3-39 CCCGAGTGTGTG 1698. 4 CTCGGGGACTGAC 1699. GGAGCAGC 1700. DMSO 3 0 1 TGTGCGTGTGG AAGCCGG TCTTCCAG GGCC OT3-40 CTGGAGTGAGTG 1701. 4 CCCCGACCAAAGC 1702. CTGGCAGC 1703. DMSO 1 2 1 TGTGTGTGTGG AGGAGCA CTCTGGAT GGGG OT3-41 GTTTCATGAGTG 1704. 4 Not optimized 0 3 1 TGTGCGTGGGG OT3-42 TATGTGTGCGTG 1705. 4 ATTTCAGAGCCCC 1706. AGGCCGCG 1707. DMSO 1 2 1 TGTGCGTGTGG GGGGAAA GTGTTATG GTTA OT3-43 TATGTGTGTGTG 1708. 4 GCCAGTGGCTTAG 1709. TGACATAT 1710. DMSO 2 1 1 TGTGCGTGGGG TGTCTTTGTGT TTTCCTGG GCCATGGG T OT3-44 TCTGTGTGTGTG 1711. 4 TGCCAGAAGAACA 1712. CCATGCTG 1713. DMSO 3 1 0 TGTGCGTGGGG TGGGCCAGA ACATCATA TACTGGGA AGC OT3-45 TCTGTGTGTGTG 1714. 4 GCGTGTCTCTGTG 1715. CCAGGCTG 1716. DMSO 3 1 0 TGTGCGTGTGG TGCGTGC GGCACACA GGTT OT3-46 TGAGCGTGAGTG 1717. 4 Not optimized 2 2 0 TGAGCGTGTGG OT3-47 TGTCTTTGAGTG 1718. 4 TGCCCAGTCCAAT 1719. AGGATGAG 1720. DMSO 2 2 0 TGTGCGTGTGG ATTTCAGCAGCT TTCATGTC CTTTGTGG GG OT3-48 TTTGTGTGTGTG 1721. 4 GGGTGAAAATTTG 1722. AATGACTC 1723. DMSO 2 2 0 TGTGCGTGTGG GTACTGTTAGCTG ATTCCCTG T GGTATCTC CCA OT3-49 AAGGCGTGTGTG 1724. 5 TGCCCCATCAATC 1725. CAAGGTCG 1726. DMSO 1 2 2 TGTGCGTGTGG ACCTCGGC GCAGGGCA GTGA OT3-50 AATTCGTGTGTG 1727. 5 GCCTCCTCTGCCG 1728. TGAGAGTT 1729. DMSO 1 2 2 TGTGCGTGGGG CTGGTAA CCTGTTGC TCCACACT OT3-51 ATGGTGTGTGTG 1730. 5 Not optimized 2 2 1 TGTGCGTGTGG OT3-52 CACGTGTGTGTG 1731. 5 GCCACCAAAATAG 1732. ACATGCAT 1733. DMSO 3 0 2 TGTGCGTGTGG CCAGCGT CTGTGTGT GCGT OT3-53 GAAATTTGAGTG 1734. 5 ACAGACTGACCCT 1735. TGTATCTT 1736. DMSO 2 1 2 TGTGCGTGTGG TGAAAAATACCAG TCTTGCCA T ATGGTTTT CCC OT3-54 TAAGTGTGTGTG 1737. 5 AGCCAAATTTCTC 1738. TCCTGGAG 1739. DMSO 3 1 1 TGTGCGTGTGG AACAGCAGCACT AGCAGGCA TTTTTGT OT3-55 TATATGTGTGTG 1740. 5 ACCTCCTTGTGCT 1741. GGCGGGAA 1742. DMSO 2 1 2 TGTGCGTGGGG GCCTGGC GGTAACCC TGGG OT3-56 TATCTGTGTGTG 1743. 5 CACAAAGCTCTAC 1744. TGATCCGA 1745. DMSO 3 1 1 TGTGCGTGTGG CTTTCCAGTAGTG TGGTTGTT T CACAGCT OT3-57 TTTATGTGTGTG 1746. 5 TGTGGGGATTACC 1747. ACGCACAA 1748. DMSO 2 2 1 TGTGCGTGTGG TGCCTGGC AAATGCCC TTGTCA OT3-58 TTTTTGTGTGTG 1749. 5 TGAGGCAGACCAG 1750. GCCCGAGC 1751. DMSO 2 3 0 TGTGCGTGGGG TCATCCAGC ACAGTGTA GGGC OT3-59 AAAAATTGTGTG 1752. 6 ATTAGCTGGGCGT 1753. ACTGCATC 1754. DMSO 2 1 3 TGTGCGTGGGG GGCGGAG TCATCTCA GGCAGCT OT3-60 ACAATGTGTGTG 1755. 6 TGAAGCAGAAGGA 1756. TCAGCTTC 1757. DMSO 4 0 2 TGTGCGTGTGG GTGGAGAAGGA ACATCTGT TTCAGTTC AGT OT3-61 ATGTGGTGTGTG 1758. 6 TGGTGGAGTGTGT 1759. AGAGCAGA 1760. DMSO 1 3 2 TGTGCGTGTGG GTGTGGT AAGAGAGT GCCCA OT3-62 CAAAATTGTGTG 1761. 6 GCCCCTGTACGTC 1762. TGCACAAG 1763. DMSO 3 1 2 TGTGCGTGTGG CTGACAGC CCACTTAG CCTCTCT OT3-63 CCCTGGTGTGTG 1764. 6 AGCGCAGGTAAAC 1765. TCTCTCGC 1766. DMSO 3 1 2 TGTGCGTGTGG AGGCCCA CCCGTTTC CTTGT OT3-64 TCCGCTTGTGTG 1767. 6 ATGGGTGCCAGGT 1768. ACAGCAGG 1769. DMSO 2 3 1 TGTGCGTGGGG ACCACGC AAGGAGCC GCAG OT3-65 TCCTCGTGTGTG 1770. 6 CGGGCGGGTGGAC 1771. AGGAGGTC 1772. DMSO 2 3 1 TGTGCGTGGGG AGATGAG TCGAGCCA GGGG OT3-66 TTAAGGTGGGTG 1773. 6 TCAACCTAGTGAA 1774. GTCTATAT 1775. DMSO 1 2 3 TGTGCGTGGGG CACAGACCACTGA ACAGCCCA CAACCTCA TGT OT3-67 TTATATTGTGTG 1776. 6 GCCAGGGCCAGTG 1777. TGTCATTT 1778. DMSO 2 4 0 TGTGCGTGGGG GATTGCT CTTAGTAT GTCAGCCG GA OT3-68 TTGAGGAGAGTG 1779. 6 GAGCCCCACCGGT 1780. GCCAGAGC 1781. DMSO 1 3 2 TGTGCGTGAGG TCAGTCC TACCCACT CGCC 1782. 1783 1784. Target 4 GAGTCCGAGCAG 1785. 0 GGAGCAGCTGGTC 1786. GGGAAGGG 1787. DMSO AAGAAGAAGGG AGAGGGG GGACACTG GGGA OT4-1 GAGTTAGAGCAG 1788. 2 TCTCTCCTTCAAC 1789. ATCTGCAC 1790. DMSO 0 1 1 AGGAAGAAAGG TCATGACCAGCT ATGTATGT ACAGGAGT CAT OT4-2 AAGTCAGAGGAG 1791. 3 AAGACAGAGGAGAA TGGGGAATCTCCA 1792. AGGGTGTA 1793. DMSO 2 1 1 AAGAAGAAGGG GAAGAAGGG (SEQ AAGAACCCCC CTGTGGGA ID NO: 2229) ACTTTGCA OT4-3 AAGTCCGAGGAG 1794. 3 GATGGCCCCACTG 1795. ACTTCGTA 1796. DMSO 1 0 2 AGGAAGAAAGG AGCACGT GAGCCTTA AACATGTG GC OT4-4 AAGTCTGAGCAC 1797. 3 AGGATTAATGTTT 1798. TCAAACAA 1799. 1M 1 0 2 AAGAAGAATGG AAAGTCACTGGTG GGTGCAGA betaine, G TACAGCA TD OT4-5 ACGTCTGAGCAG 1800. 3 TCCAAGCCACTGG 1801. TGCTCTGT 1802. DMSO 0 1 2 AAGAAGAATGG TTTCTCAGTCA GGATCATA TTTTGGGG GA OT4-6 GACTCCTAGCAA 1803. 3 ACTTTCAGAGCTT 1804. CCCACGCT 1805. DMSO 1 1 1 AAGAAGAATGG GGGGCAGGT GAAGTGCA ATGGC OT4-7 GAGACTGAGAAG 1806. 3 CAAAGCATGCCTT 1807. GGCTCTTC 1808. 1M 1 1 1 AAGAAGAAAGG TCAGCCG GATTTGGC betaine, ACCT TD OT4-8 GAGCCGGAGCAG 1809. 3 Not optimized 1 0 2 AAGAAGGAGGG OT4-9 GAGCCTGAGCAG 1810. 3 GGACTCCCTGCAG 1811. AGGAACAC 1812. 72C 0 0 3 AAGGAGAAGGG CTCCAGC AGGCCAGG Anneal, CTGG 6% DMSO OT4-10 GAGGCCGAGCAG 1813. 3 CCCTTTAGGCACC 1814. CCGACCTT 1815. DMSO 0 1 2 AAGAAAGACGG TTCCCCA CATCCCTC CTGG OT4-11 GAGTAAGAGAAG 1816. 3 TGATTCTGCCTTA 1817. TGGGCTCT 1818. DMSO 0 3 0 AAGAAGAAGGG GAGTCCCAGGT GTGTCCCT ACCCA OT4-12 GAGTAGGAGGAG 1819. 3 Not optimized 2 1 0 AAGAAGAAAGG OT4-13 GAGTCCGGGAAG 1820. 3 AGGCAGGAGAGCA 1821. ACCCTGAC 1822. DMSO 0 1 2 GAGAAGAAAGG AGCAGGT TACTGACT GACCGCT OT4-14 GATTCCTACCAG 1823. 3 CTCCCCATTGCGA 1824. AGAGGCAT 1825. DMSO 1 2 0 AAGAAGAATGG CCCGAGG TGACTTGG AGCACCT OT4-15 GCGACAGAGCAG 1826. 3 CTGGAGCCCAGCA 1827. CCTCAGGG 1828. DMSO 1 2 0 AAGAAGAAGGG GGAAGGC AGGGGGCC TGAT OT4-16 AAATCCAACCAG 1829. 4 ACTGTGGGCGTTG 1830. AGGTCGGT 1831. DMSO 1 0 3 AAGAAGAAAGG TCCCCAC GCAGGGTT TAAGGA OT4-17 AAGTCTGAGGAC 1832. 4 GGCGCTCCCTTTT 1833. CGTCACCC 1834. DMSO 2 0 2 AAGAAGAATGG TCCCTTTGT ATCGTCTC GTGGA OT4-18 AAGTTGGAGCAG 1835. 4 TGCCATCTATAGC 1836. GCATCTTG 1837. DMSO 1 0 3 GAGAAGAAGGG AGCCCCCT CTAACCGT ACTTCTTC TGA OT4-19 AATACAGAGCAG 1838. 4 GTGGAGACGCTAA 1839. GCTCCTGG 1840. DMSO 1 2 1 AAGAAGAATGG ACCTGTGAGGT CCTCTTCC TACAGC OT4-20 AGGTACTAGCAG 1841. 4 CCGAACTTCTGCT 1842. CCAAGTCA 1843. DMSO 0 2 2 AAGAAGAAAGG GAGCTTGATGC ATGGGCAA CAAGGGA OT4-21 AGGTGCTAGCAG 1844. 4 Not optimized 1 1 2 AAGAAGAAGGG OT4-22 AGGTGGGAGCAG 1845. 4 TGCCCCCAAGACC 1846. ATGGCAGG 1847. DMSO 2 0 2 AAGAAGAAGGG TTTCTCC CAGAGGAG GAAC OT4-23 CAAACGGAGCAG 1848. 4 GGGTGGGGCCATT 1849. CTGGGGCC 1850. DMSO 3 0 1 AAGAAGAAAGG GTGGGTT AGGGTTTC TGCC OT4-24 CACTCTGAGGAG 1851. 4 TGGAGAACATGAG 1852. TCCTTCTG 1853. DMSO 3 0 1 AAGAAGAAAGG AGGCTTGCAA TAGGCAAT GGGAACAA OT4-25 CAGTCATGGCAG 1854. 4 GCCACATGGTAGA 1055. GGCAGATT 1856. 1M 1 2 1 AAGAAGAAAGG AGTCGGC TCCCCCAT betaine, GCTG TD OT4-26 CCGTCCCAGCAG 1857. 4 TGTACACCCCAAG 1858. AAGGGGAG 1859. DMSO 3 1 0 TAGAAGAATGG TCCTCCC TGTGCAAG CCTC OT4-27 GTCTGCGATCAG 1860. 4 AGGTCTGGCTAGA 1861. AGTCCAAC 1862. DMSO 3 1 0 AAGAAGAAAGG GATGCAGCA ACTCAGGT GAGACCCT OT4-28 TAATCCAATCAG 1863. 4 CCAAGAGGACCCA 1864. GGGTATGG 1865. DMSO 0 2 2 AAGAAGAAGGG GCTGTTGGA AATTCTGG ATTAGCAG AGC OT4-29 TATACGGAGCAG 1866. 4 ACCATCTCTTCAT 1867. ACACTGTG 1868. DMSO 2 2 0 AAGAAGAATGG TGATGAGTCCCAA AGTATGCT TGGCGT OT4-30 ACTTCCCTGCAG 1869. 5 GGCTGCGGGGAGA 1870. TCGGATGC 1871. DMSO 2 2 1 AAGAAGAAAGG TGAGCTC TTTTCCAC AGGGCT OT4-31 AGGACTGGGCAG 1872. 5 TCTTCCAGGAGGG 1873. CCAATCCT 1874. DMSO 1 0 4 AAGAAGAAGGG CAGCTCC GAGCTCCT ACAAGGCT OT4-32 AGGTTGGAGAAG 1875. 5 GAGCTGCACTGGA 1876. TGCTGGTT 1877. DMSO 1 1 3 AAGAAGAAGGG TGGCACT AAGGGGTG TTTTGGA OT4-33 AGTTCAGAGCAG 1878. 5 TCTGGGAAGGTGA 1879. TGGGGGAC 1880. DMSO 0 2 3 GAGAAGAATGG GGAGGCCA AATGGAAA AGCAATGA OT4-34 ATGACACAGCAG 1881. 5 CTTGCTCCCAGCC 1882. AGCCCTTG 1883. DMSO 3 1 1 AAGAAGAAGGG TGACCCC CCATGCAG GACC OT4-35 ATGACAGAGAAG 1884. 5 GGGATTTTTATCT 1885. AACCACAG 1886. DMSO 2 2 1 AAGAAGAAAGG GTTGGGTGCGAA ATGTACCC TCAAAGCT OT4-36 CCGCCCCTGCAG 1887. 5 ACCCATCAGGACC 1888. TCTGGAAC 1889. 72C 3 1 1 AAGAAGAACGG GCAGCAC CTGGGAGG Anneal, CGGA 3% DMSO OT4-37 GCAGGAGAGCAG 1890. 5 CGTCCCTCACAGC 1891. CCTCCTTG 1892. DMSO 1 3 1 AAGAAGAAAGG CAGCCTC GGCCTGGG GTTC OT4-38 GTTCAAGAGCAG 1893. 5 CCCTCTGCAAGGT 1894. AGATGTTC 1895. DMSO 1 3 1 AAGAAGAATGG GGAGTCTCC TGTCCCCA GGCCT OT4-39 GTTTTGAAGCAG 1896. 5 GGCTTCCACTGCT 1897. TGCCGCTC 1898. DMSO 2 1 2 AAGAAGAAAGG GAAGGCCT CACATACC CTCC OT4-40 TATGGCAAGCAG 1899. 5 AGCATTGCCTGTC 1900. AGCACCTA 1901. DMSO 1 3 1 AAGAAGAAAGG GGGTGATGT TTGGACAC TGGTTCTC T OT4-41 TGGTGGGATCAG 1902. 5 TCTAGAGCAGGGG 1903. TGGAGATG 1909. DMSO 2 2 1 AAGAAGAAAGG CACAATGC GAGCCTGG TGGGA OT4-42 ACCCACGGGCAG 1905. 6 GGTCTCAGAAAAT 1906. CCCACAGA 1907. DMSO 1 2 3 AAGAAGAAGGG GGAGAGAAAGCAC AACCTGGG G CCCT OT4-43 ACTCCTGATCAG 1908. 6 GGTTGCTGATACC 1909. TGGGTCCT 1910. DMSO 0 3 3 AAGAAGAAGGG AAAACGTTTGCCT CTCCACCT CTGCA OT4-44 ACTGATGAGCAG 1911. 6 ACTCTCCTTAAGT 1912. CAGAATCT 1913. DMSO 0 4 2 AAGAAGAAAGG ACTGATATGGCTG TGCTCTGT T TGCCCA OT4-45 ATTTTAGTGCAG 1914. 6 Not optimized 2 2 2 AAGAAGAAAGG OT4-46 ATTTTAGTGCAG 1915. 6 Not optimized 2 2 2 AAGAAGAAAGG OT4-47 CCATGGCAGCAG 1916. 6 CAATGCCTGCAGT 1917. TCCCAAGA 1918. DMSO A 1 1 AAGAAGAAGGG CCTCAGGA GAAAACTC TGTCCTGA CA OT4-48 CCATTACAGCAG 1919. 6 GCATTGGCTGCCC 1920. TGGCTGTG 1921. DMSO 2 2 2 AAGAAGAAGGG AGGGAAA CTGGGCTG TGTT OT4-49 CGAGGCGGGCAG 1922. 6 CCACAAGCCTCAG 1923. ACAGGTGC 1924. DMSO 2 1 3 AAGAAGAAAGG CCTACCCG CAAAACAC TGCCT OT4-50 TCATTGCAGCAG 1925. 6 TCATTGCAGCAGAA GCCTCTTGCAAAT 1926. CGATCAGT 1927. DMSO 2 / 1 2 / 3 2 AAGAAGAAAGG GAAGAAAGG GAGACTCCTTTT CCCCTGGC TCATTGTAGCAGAA GTCC GAAGAAAGG (SEQ ID NO: 2230) OT4-51 TCTCCAGGGCAG 1928. 6 TCCCAGAATCTGC 1929. AGGGGTTT 1930. DMSO 0 4 2 AAGAAGAAAGG CTCCGCA CCAGGCAC ATGGG 1931. 1932. 1933. Target 5 GTCATCTTAGTC 1934. 0 TCCTAAAAATCAG 1935. AAAGTGTT 1936. DMSO ATTACCTGAGG TTTTGAGATTTAC AGCCAACA TTCC TACAGAAG TCAGGA OT5-1 GGTATCTAAGTC 1937. 3 GGTATCTAAGTCAT ACATCTGGGGAAA 1938. TGTCTGAG 1939. DMSO 1 / 2 1 1 ATTACCTGTGG TACCTGTGG (SEQ GCAAAAGTCAACA TATCTAGG ID NO: 2231) CTAAAAGT GGTATCTAAGTCAA GGT TACCTGTGG (SEQ ID NO: 2232) OT5-2 GTAATATTAGTC 1940. 3 ACGATCTTGCTTC 1941. AGTGCTTT 1942. DMSO 0 3 0 ATTACCGGTGG ATTTCCCTGTACA GTGAACTG AAAAGCAA OT5-3 GTAATCTGAGTC 1943. 3 GCACCTTGGTGCT 1944. ACA ATTTCCTGGGG GCTAAATGCC GGGCAACT 1945. DMSO 1 2 0 GAACAGGC ATGAATGG OT5-4 GTCATCCTAGTC 1946. 3 AACTGTCCTGCAT 1947. GGTGCACC 1948. DMSO 1 1 1 ATTTACTGGGG CCCCGCC TGGATCCA CCCA OT5-5 GTCATCCTAGTG 1949. 3 Not optimized 1950. 1951. 1 1 1 CTTACCTGAGG OT5-6 GTCATCTGAGGC 1952. 3 CATCACCCTCCAC 1953. ACCACTGC 1954. 72C 0 3 0 ATTAACTGGGG CAGGCCC TGCAGGCT Anneal, CCAG 3% DMSO OT5-7 AATATGTTAGTC 1955. 4 Not optimized 2 0 2 ATTACCTGAGG OT5-8 ATAAACGTAGTC 1956. 4 CCTGACCCGTGGT 1957. TGGTGCGT 1958. 72C 1 2 1 ATTACCTGGGG TCCCGAC GGTGTGTG Anneal, TGGT 3% DMSO OT5-9 ATCATCATCGTC 1959. 4 TGGGAACATTGGA 1960. CCATGTGA 1961. DMSO 1 1 2 ATTATCTGGGG GAAGTTTCCTGA CTACTGGG CTGCCC OT5-10 ATCATTTTACTC 1962. 4 AGCCTTGGCAAGC 1963. GGTTCTCT 1964. DMSO 1 0 3 ATTACTTGTGG AACTCCCT CTCTCAGA AAAGAAAG AGG OT5-11 ATCATTTTAGTC 1965. 4 GGCAGCGGACTTC 1966. GCCAGAGG 1967. DMSO 1 0 3 ATCTCCTGTGG AGAGCCA CTCTCAGC AGTGC OT5-12 CACAGCTTAGTC 1968. 4 CCAGCCTGGTCAA 1969. ACTGTGCC 1970. DMSO 2 1 1 ATCACCTGGGG TATGGCA CAGCCCCA TATT OT5-13 CCCAGCTTAGTC 1971. 4 ATGCCAACACTCG 1972. CGGGTTGT 1973. DMSO 2 1 1 ATTAGCTGTGG AGGGGCC GGCACCGG GTTA OT5-14 CTCACCTTTGTC 1974. 4 TTGCTCTAGTGGG 1975. AGAGTTCA 1976. DMSO 3 0 1 ATTTCCTGAGG GAGGGGG GGCATGAA AAGAAGCA ACA OT5-15 CTCATTTTATTC 1977. 4 AGCTGAAGATAGC 1978. TGCAATTT 1979. DMSO 1 1 2 ATTGCCTGGGG AGTGTTTAAGCCT GAGGGGCT CTCTTCA OT5-16 CTCTCCTTAGTC 1980. 4 AGTCACTGGAGTA 1981. TGCCAGCC 1982. DMSO 2 0 2 ACTACCTGAGG AGCCTGCCT AAAAGTTG TTAGTGTG T OT5-17 CTTATCTCTGTC 1983. 4 GGGTCTCCCTCAG 1984. TGTGTGGT 1985. DMSO 2 0 2 ATTACCTGGGG TGCCCTG AGGGAGCA AAACGACA OT5-18 GACAGCTCCGTC 1986. 4 TGGGGGCTGTTAA 1987. TGACCACA 1988. DMSO 1 2 1 ATTACCTGGGG GAGGCACA CACACCCC CACG OT5-19 GCCACCTCAGTC 1989. 4 TCAAAACAGATTG 1990. TGTGTTTT 1991. DMSO 1 0 3 ATTAGCTGGGG ACCAAGGCCAAAT TAAGCTGC ACCCCAGG OT5-20 GGAATCTTACTC 1992. 4 TCTGGCACCAGGA 1993. GCACGCAG 1994. DMSO 1 2 1 ATTACTTGGGG CTGATTGTACA CTGACTCC CAGA OT5-21 GTGGCCTCAGTC 1995. 4 Not optimized 1 0 3 ATTACCTGGGG OT5-22 GTTGTTTTAGTG 1996. 4 AGCATCTGTGATA 1997. ACCAGGGC 1998. DMSO 1 0 3 ATTACCTGAGG CCCTACCTGTCT TGCCACAG AGTC OT5-23 TACATCTTAGTC 1999. 4 TAGTCTTGTTGCC 2000. CTCGGCCC 2001. DMSO 1 2 1 CTCACCTGTGG CAGGCTG CTGAGAGT TCAT OT5-24 TCCATCTCACTC 2002. 4 TCCATCTCACTCAT CTGCAACCAGGGC 2003. GAGCAGCA 2004. DMSO 1 1 2 ATTACCTGAGG TACCTGAGG (SEQ CCTTACC GCAAAGCC ID NO: 2233) ACCG TCCATCTCACTCAT TACCTGATG (SEQ ID NO: 2234) OT5-25 TTCATCCTAGTC 2005. 4 GCCTGGAGAGCAA 2006. AGCCGAGA 2007. DMSO 1 1 2 AACACCTGGGG GCCTGGG CAATCTGC CCCG OT5-26 TTTATATTAGTG 2008. 4 TTTATATTAGTGAT AGTGAAACAAACA 2009. GGCAGGTC 2010. No 1 2 1 ATTACCTGTGG TACCTGCGG (SEQ AGCAGCAGTCTGA TGACCAGT DMSO ID NO: 2235) GGGG TD OT5-27 AACGTGTAAGTC 2011. 5 AGGCTCAGAGAGG 2012. TGAGTAGA 2013. DMSO 3 0 2 ATTACCTGAGG TAAGCAATGGA CAGAAATG TTACCGGT GTT OT5-28 AAGATCACAGTC 2014. 5 TCAGAGATGTTAA 2015. AGTGAACC 2016. DMSO 3 0 2 ATTACCTGGGG AGCCTTGGTGGG AAGGGAAT GGGGGA OT5-29 AGAATATTAGTC 2017. 5 TGTGCTTTCTGGG 2018. CACCTCAG 2019. DMSO 0 4 1 CTTACCTGGGG GTAGTGGCA CCCTGTAG TCCTGG OT5-30 AGCAGATTAGTG 2020. 5 CCATTGGGTGACT 2021. GCCACTGT 2022. 1M 1 3 1 ATTACCTGGGG GAATGCACA CCCCAGCC betaine, TATT TD OT5-31 AGTAGCTTAGTG 2023. 5 ACCAAGAAAGTGA 2024. TGAGATGG 2025. DMSO 1 2 2 ATTACCTGGGG AAAGGAAACCC CATACGAT TTACCCA OT5-32 CACGGCTTACTC 2026. 5 AGGGTGGGGACTG 2027. TGGCATCA 2028. DMSO 3 1 1 ATTACCTGGGG AAAGGAGCT CTCAGAGA TTGGAACA CA OT5-33 CATATGTTAGGC 2029. 5 ACCAGTGCTGTGT 2030. TCCTATGG 2031. DMSO 3 1 1 ATTACCTGGGG GACCTTGGA GAGGGGAG GCTTCT OT5-34 CATTTCTTAGTC 2032. 5 CCAGGTGTGGTGG 2033. GCATACGG 2039. 68C, 9 0 1 ATTTCCTGAGG TTCATGAC CAGTAGAA 3% TGAGCC DMSO OT5-35 TGCAGCTAACTC 2035. 5 CAGGCGCTGGGTT 2036. CCTTCCTG 2037. DMSO 2 3 0 ATTACCTGGGG CTTAGCCT GGCCCCAT GGTG OT5-36 TTGCTTTTAGTT 2038. 5 TGGGGTCCAAGAT 2039. TGAAACTG 2040. DMSO 1 2 2 ATTACCTGGGG GTCCCCT CTTGATGA GGTGTGGA OT5-37 AACTTGAAAGTC 2041. 6 GCTGGGCTTGGTG 2042. ACTTGCAA 2043. DMSO 5 0 1 ATTACCTGTGG GTATATGC AGCTGATA ACTGACTG A OT5-38 AAGGTCACAGTC 2044. 6 AGTTGGTGTCACT 2045. CGCAGCGC 2046. DMSO 3 0 3 ATTACCTGGGG GACAATGGGA ACGAGTTC ATCA OT5-39 AATGTCTTCATC 2047. 6 AGAGGAGGCACAA 2048. GGCTGGGG 2049. DMSO 1 1 4 ATTACCTGAGG TTCAACCCCT AGGCCTCA CAAT OT5-40 AGATGCTTGGTC 2050. 6 GGGAAAGTTTGGG 2051. AGGACAAG 2052. DMSO 1 3 2 ATTACCTGTGG AAAGTCAGCA CTACCCCA CACC OT5-41 AGTAGATTAGTT 2053. 6 TGGTGCATCAAAG 2054. TCATTCCA 2055. DMSO 0 3 3 ATTACCTGGGG GGTTGCTTCT GCACGCCG GGAG OT5-42 AGTAGGTTAGTA 2056. 6 CCCAGGCTGCCCA 2057. TGGAGTAA 2058. DMSO 1 3 2 ATTACCTGGGG TCACACT GTATACCT TGGGGACC T OT5-43 CAAATGAGAGTC 2059. 6 TCAGTGCCCCTGG 2060. TGTGCAAA 2061. DMSO 9 2 0 ATTACCTGAGG GTCCTCA TACCTAGC ACGGTGC OT5-44 CATGTCTGAATC 2062. 6 AGCACTCCCTTTT 2063. ACTGAAGT 2069. DMSO 2 1 3 ATTACCTGAGG GAATTTTGGTGCT CCAGCCTC TTCCATTT CA OT5-45 CCTGACTTGGTC 2065. 6 GAAACCGGTCCCT 2066. GGGGAGTA 2067. DMSO 2 0 4 ATTACCTGTGG GGTGCCA GAGGGTAG TGTTGCC OT5-46 CGTGCATTAGTC 2068. 6 TTGCGGGTCCCTG 2069. AGGTGCCG 2070. DMSO 1 2 3 ATTACCTGAGG TGGAGTC TGTTGTGC CCAA Target 6 GGAATCCCTTCT 2071. 0 GCCCTACATCTGC 2072. GGGCCGGG 2073. DMSO GCAGCACCTGG TCTCCCTCCA AAAGAGTT GCTG OT6-1 GGAACCCCGTCT 2074. 2 TTGGAGTGTGGCC 2075. ACCTCTCT 2076. DMSO 0 1 1 GCAGCACCAGG CGGGTTG TTCTCTGC CTCACTGT OT6-2 GGAACACCTTCT 2077. 3 CACACCATGCTGA 2078. GCAGTACG 2079. DMSO 1 1 1 GCAGCTCCAGG TCCAGGC GAAGCACG AAGC OT6-3 GGAAGCTCTGCT 2080. 3 CTCCAGGGCTCGC 2081. CTGGGCTC 2082. DMSO 0 2 1 GCAGCACCTGG TGTCCAC TGCTGGTT CCCC OT6-4 GGAATATCTTCT 2083. 3 CTGTGGTAGCCGT 2084. CCCCATAC 2085. DMSO 0 2 1 GCAGCCCCAGG GGCCAGG CACCTCTC CGGGA OT6-5 GGAATCACTTTT 2086. 3 GGTGGCGGGACTT 2087. CCAGCGTG 2088. 1M 0 1 2 ACAGCACCAGG GAATGAG TTTCCAAG betaine, GGAT TD OT6-6 GGAATCCCCTCT 2089. 3 GGAATCCCCTCTCC CCAGAGGTGGGGC 2090. TTTCCACA 2091. DMSO 1 1 1 / 2 CCAGCCCCTGG AGCCCCTGG (SEQ CCTGTGA CTCAGTTC ID NO: 2236) TGCAGGA GGAATCCCCTCTCC AGCCTCTGG(SEQ ID NO: 2237) OT6-7 GGAATCTCTTCT 2092. 3 GGAATCTCTTCCTT TGTGACTGGTTGT 2093. GCAGTGTT 2094. 1M 0 1 5 TCAGCATCTGG GGCATCTGG(SEQ CCTGCTTTCCT TTGTGGTG betaine, ID NO: 2238) ATGGGCA TD OT6-8 GGAATTGCTTCT 2095. 3 CTGGCCAAGGGGT 2096. TGGGACCC 2097. DMSO 1 0 2 GCAGCGCCAGG GAGTGGG CAGCAGCC AATG OT6-9 GGACTCCCCTCT 2098. 3 ACGGTGTGCTGGC 2099. ACAGTGCT 2100. DMSO 1 1 1 GCAGCAGCTGG TGCTCTT GACCGTGC TGGG OT6-10 GGAGTCCCTCCT 2101. 3 TGGTTTGGGCCTC 2102. TGCCTCCC 2103. DMSO 0 0 3 ACAGCACCAGG AGGGATGG ACAAAAAT GTCTACCT OT6-11 GGAGTCCCTCCT 2104. 3 TGGTTTGGGCCTC 2105. ACCCCTTA 2106. DMSO 0 0 3 ACAGCACCAGG AGGGATGG TCCCAGAA CCCATGA OT6-12 GGCATCCATTCT 2107. 3 TCCAAGTCAGCGA 2108. TGGGAGCT 2109. DMSO 0 3 0 GCAGCCCCTGG TGAGGGCT GTTCCTTT TTGGCCA OT6-13 GGCTTCCCTTCT 2110. 3 CACCCCTCTCAGC 2111. GCTAGAGG 2112. DMSO 1 2 0 GCAGCCCCAGG TTCCCAA GTCTGCTG CCTT OT6-14 TGAATCCCATCT 2113. 3 AGACCCCTTGGCC 2114. CTTGCTCT 2115. DMSO 2 1 0 CCAGCACCAGG AAGCACA CACCCCGC CTCC OT6-15 AAAATACCTTCT 2116. 4 ACATGTGGGAGGC 2117. TCTCACTT 2118. DMSO 0 1 3 GCAGTACCAGG GGACAGA TGCTGTTA CCGATGTC G OT6-16 AAAATCCCTTCT 2119. 4 GGACGACTGTGCC 2120. AGTGCCCA 2121. 72C 0 1 3 TCAACACCTGG TGGGACA GAGTGTTG Anneal, TAACTGCT 3% DMSO OT6-17 ACACTCCCTCCT 2122. 4 GGAGAGCTCAGCG 2123. CAGCGTGG 2124. DMSO 1 1 2 GCAGCACCTGG CCAGGTC CCCGTGGG AATA OT6-18 ACCATCCCTCCT 2125. 4 GCTGAAGTGCTCT 2126. ACCCCACT 2127. DMSO 1 1 2 GCAGCACCAGG GGGGTGCT GTGGATGA ATTGGTAC C OT6-19 AGAGGCCCCTCT 2128. 4 TCGGGGTGCACAT 2129. TTGCCTCG 2130. DMSO 0 1 3 GCAGCACCAGG GGCCATC CAGGGGAA GCAG OT6-20 AGGATCCCTTGT 2131. 4 CTCGTGGGAGGCC 2132. AGCCACCA 2133. DMSO 2 0 2 GCAGCTCCTGG AACACCT ACACATAC CAGGCT OT6-21 CCACTCCTTTCT 2134. 4 GCATGCCTTTAAT 2135. AGGATTTC 2136. DMSO 2 1 1 GCAGCACCAGG CCCGGCT AGAGTGAT GGGGCT OT6-22 GAAGGCCCTTCA 2137. 4 CGCCCAGCCACAA 2138. GCAAATTT 2139. DMSO 1 1 2 GCAGCACCTGG AGTGCAT CTGCACCT ACTCTAGG CCT OT6-23 GATATCCCTTCT 2140. 4 AGCTCACAAGAAT 2141. GCAGTCAC 2142. DMSO 1 1 2 GTATCACCTGG TGGAGGTAACAGT CCTTCACT GCCTGT OT6-24 GGGTCCGCTTCT 2143. 4 AAACTGGGCTGGG 2144. GGGGCTAA 2145. DMSO 2 0 2 GCAGCACCTGG CTTCCGG GGCATTGT CAGACCC OT6-25 GTCTCCCCTTCT 2146. 4 GCAGGTAGGCAGT 2147. TCTCCTGC 2148. 1M 1 2 1 GCAGCACCAGG CTGGGGC CTCAGCCT betaine, CCCA TD OT6-26 GTCTCCCCTTCT 2149. 4 GCAGGTAGGCAGT 2150. TCTCCTGC 2151. 1M 1 2 1 GCAGCACCAGG CTGGGGC CTCAGCCT betaine, CCCA TD OT6-27 GTCTCCCCTTCT 2152. 4 GCAGGTAGGCAGT 2153. TCTCCTGC 2154. 1M 1 2 1 GCAGCACCAGG CTGGGGC CTCAGCCT betaine, CCCA TD OT6-28 TCATTCCCGTCT 2155. 4 GCTCTGGGGTAGA 2156. GGCCTGTC 2157. DMSO 2 2 0 GCAGCACCCGG AGGAGGC AACCAACC AACC OT6-29 TGCACCCCTCCT 2158. 4 TGACATGTTGTGT 2159. AAATCCTG 2160. DMSO 0 2 2 GCAGCACCAGG GCTGGGC CAGCCTCC CCTT OT6-30 TGCATACCCTCT 2161. 4 TCCTGGTGAGATC 2162. TCCTCCCC 2163. DMSO 0 3 1 GCAGCACCAGG GTCCACAGGA ACTCAGCC TCCC OT6-31 TGCATGGCTTCT 2164. 4 TCCTAATCCAAGT 2165. AGGGACCA 2166. DMSO 2 2 0 GCAGCACCAGG CCTTTGTTCAGAC GCCACTAC A CCTTCA OT6-32 AATATTCCCTCT 2167. 5 GGGACACCAGTTC 2168. GGGGGAGA 2169. DMSO 1 0 4 GCAGCACCAGG CTTCCAT TTGGAGTT CCCC OT6-33 ACCATTTCTTCT 2170. 5 ACACCACTATCAA 2171. TCTGCCTG 2172. DMSO 1 1 3 GCAGCACCTGG GGCAGAGTAGGT GGGTGCTT TCCC OT6-34 AGCTCCCATTCT 2173. 5 CTGGGAGCGGAGG 2174. GCCCCGAC 2175. DMSO 1 2 2 GCAGCACCCGG GAAGTGC AGATGAGG CCTC OT6-35 CAGATTCCTGCT 2176. 5 CAGATTACTGCTGC CGGGTCTCGGAAT 2177. ACCCAGGA 2178. DMSO 1 2 3 GCAGCACCGGG AGCACCGGG (SEQ GCCTCCA ATTGCCAC ID NO: 2239) CCCC OT6-36 CCAAGAGCTTCT 2179. 5 TTGCTGTGGTCCC 2180. GCAGACAC 2181. DMSO 3 2 0 GCAGCACCTGG GGTGGTG TAGAGCCC GCCC OT6-37 CCCAGCCCTGCT 2182. 5 GGTGTGGTGACAG 2183. ACCTGCGT 2184. DMSO 2 3 0 GCAGCACCCGG GTCGGGT CTCTGTGC TGCA OT6-38 CCCCTCCCTCCT 2185. 5 CTCCCAGGACAGT 2186. CCTGGCCC 2187. DMSO 2 2 1 GCAGCACCGGG GCTGGGC CATGCTGC CTG OT6-39 CTACTGACTTCT 2188. 5 TGCGTAGGTTTTG 2189. AGGGAATG 2190. DMSO 2 3 0 GCAGCACCTGG CCTCTGTGA ATGTTTTC CACCCCCT OT6-40 CTCCTCCCTCCT 2191. 5 CTCCGCAGCCACC 2192. TGCATTGA 2193. DMSO 1 3 1 GCAGCACCTGG GTTGGTA CGTACGAT GGCTCA OT6-41 TCTGTCCCTCCT 2194. 5 ACCTGCAGCATGA 2195. ACCTGAGC 2196. DMSO 2 1 2 GCAGCACCTGG ACTCTCGCA AACATGAC TCACCTGG OT6-42 ACACAAACTTCT 2197. 6 ACACAAACTTCTGC TCTCCAGTTTCTT 2198. ACCATTGG 2199. 1M 3 / 2 3 1 GCAGCACCTGG AGCACCTGG GCTCTCATGG TGAACCCA betaine, ACACAAACTTCTGC GTCA TD AGCACGTGG(SEQ ID NO: 2240) OT6-43 ACTGTCATTTCT 2200. 6 TGGGGTGGTGGTC 2202. TCAGCTAT 2202. DMSO 2 1 3 GCAGCACCTGG TTGAATCCA AACCTGGG ACTTGTGC T OT6-44 ACTTTATCTTCT 2203. 6 AGCAGCCAGTCCA 2204. CCCTTTCA 2205. DMSO 3 1 2 GCAGCACCTGG GTGTCCTG TCGAGAAC CCCAGGG OT6-45 ATCCTTTCTTCT 2206. 6 TGGACGCTGCTGG 2207. GAGGTCTC 2208. DMSO 0 3 3 GCAGCACCTGG GAGGAGA GGGCTGCT CGTG OT6-46 CACCACCGTTCT 2209. 6 AGGTTTGCACTCT 2210. TGGGGTGA 2211. DMSO 3 2 1 GCAGCACCAGG GTTGCCTGG TTGGTTGC CAGGT OT6-47 CATGTGGCTTCT 2212. 6 TCTTCCTTTGCCA 2213. TGCAGGAA 2214. DMSO 4 0 2 GCAGCACCTGG GGCAGCACA TAGCAGGT ATGAGGAG T OT6-48 CATTTTCTTTCT 2215. 6 GGACGCCTACTGC 2216. GCCCTGGC 2217. DMSO 3 0 3 GCAGCACCTGG CTGGACC AGCCCATG GTAC OT6-49 CTCTGTCCTTCT 2218. 6 AGGCAGTCATCGC 2219. GGTCCCAC 2220. DMSO 2 3 1 GCAGCACCTGG GTTGGTA CTTCCCCT ACAA OT6-50 CTGTACCCTCCT 2221. 6 Not optimized 3 1 2 GCAGCACCAGG OT6-51 TTGAGGCCGTCT 2222. 6 CCCCAGCCCCCAC 2223. CAGCCCAG 2224. DMSO 1 4 1 GCAGCACCTGG CAGTTTC GCCACAGC TTCA

Sanger Sequencing for Quantifying Frequencies of Indel Mutations

Purified PCR products used for T7EI assay were ligated into a Zero Blunt TOPO vector (Life Technologies) and transformed into chemically competent Top 10 bacterial cells. Plasmid DNAs were isolated and sequenced by the Massachusetts General Hospital (MGH) DNA Automation Core, using an M13 forward primer (5′-GTAAAACGACGGCCAG-3′) (SEQ ID NO:1059).

Restriction Digest Assay for Quantifying Specific Alterations Induced by HDR with ssODNs

PCR reactions of specific on-target sites were performed using Phusion high-fidelity DNA polymerase (New England Biolabs). The VEGF and EMX1 loci were amplified using a touchdown PCR program ((98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s)×10 cycles, (98° C., 10 s; 62° C., 15 s; 72° C., 30 s)×25 cycles), with 3% DMSO. The primers used for these PCR reactions are listed in Table E. PCR products were purified by Ampure XP beads (Agencourt) according to the manufacturer's instructions. For detection of the BamHI restriction site encoded by the ssODN donor template, 200 ng of purified PCR products were digested with BamHI at 37° C. for 45 minutes. The digested products were purified by Ampure XP beads (Agencourt), eluted in 20 ul 0.1×EB buffer and analyzed and quantified using a QIAXCEL capillary electrophoresis system.

TruSeq Library Generation and Sequencing Data Analysis

Locus-specific primers were designed to flank on-target and potential and verified off-target sites to produce PCR products ˜300 bp to 400 bps in length. Genomic DNAs from the pooled duplicate samples described above were used as templates for PCR. All PCR products were purified by Ampure XP beads (Agencourt) per the manufacturer's instructions. Purified PCR products were quantified on a QIAXCEL capillary electrophoresis system. PCR products for each locus were amplified from each of the pooled duplicate samples (described above), purified, quantified, and then pooled together in equal quantities for deep sequencing. Pooled amplicons were ligated with dual-indexed Illumina TruSeq adaptors as previously described (Fisher et al., 2011). The libraries were purified and run on a QIAXCEL capillary electrophoresis system to verify change in size following adaptor ligation. The adapter-ligated libraries were quantified by qPCR and then sequenced using Illumina MiSeq 250 bp paired-end reads performed by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. We analyzed between 75,000 and 1,270,000 (average ˜422,000) reads for each sample. The TruSeq reads were analyzed for rates of indel mutagenesis as previously described (Sander et al., 2013). Specificity ratios were calculated as the ratio of observed mutagenesis at an on-target locus to that of a particular off-target locus as determined by deep sequencing. Fold-improvements in specificity with tru-RGNs for individual off-target sites were calculated as the specificity ratio observed with tru-gRNAs to the specificity ratio for that same target with the matched full-length gRNA. As mentioned in the text, for some of the off-target sites, no indel mutations were detected with tru-gRNAs. In these cases, we used a Poisson calculator to determine with a 95% confidence that the upper limit of the actual number of mutated sequences would be three in number. We then used this upper bound to estimate the minimum fold-improvement in specificity for these off-target sites.

Example 2a Truncated gRNAs can Efficiently Direct Cas9-Mediated Genome Editing in Human Cells

To test the hypothesis that gRNAs truncated at their 5′ end might function as efficiently as their full-length counterparts, a series of progressively shorter gRNAs were initially constructed as described above for a single target site in the EGFP reporter gene, with the following sequence: 5′-GGCGAGGGCGATGCCACCTAcGG-3′ (SEQ ID NO:2241). This particular EGFP site was chosen because it was possible to make gRNAs to it with 15, 17, 19, and 20 nts of complementarity that each have a G at their 5′ end (required for efficient expression from the U6 promoter used in these experiments). Using a human cell-based reporter assay in which the frequency of RGN-induced indels could be quantified by assessing disruption of a single integrated and constitutively expressed enhanced green fluorescent protein (EGFP) gene (Example 1 and Fu et al., 2013; Reyon et al., 2012) (FIG. 2B), the abilities of these variable-length gRNAs to direct Cas9-induced indels at the target site were measured.

As noted above, gRNAs bearing longer lengths of complementarity (21, 23, and 25 nts) exhibit decreased activities relative to the standard full-length gRNA containing 20 nts of complementary sequence (FIG. 2H), a result that matches those recently reported by others (Ran et al., Cell 2013). However, gRNAs bearing 17 or 19 nts of target complementarity showed activities comparable to or higher than the full-length gRNA, while a shorter gRNA bearing only 15 nts of complementary failed to show significant activity (FIG. 2H).

To test the generality of these initial findings, full-length gRNAs and matched gRNAs bearing 18, 17 and/or 16 nts of complementarity to four additional EGFP reporter gene sites (EGFP sites #1, #2, #3, and #4; FIG. 3A) were assayed. At all four target sites, gRNAs bearing 17 and/or 18 nts of complementarity functioned as efficiently as (or, in one case, more efficiently than) their matched full-length gRNAs to induce Cas9-mediated disruption of EGFP expression (FIG. 3A). However, gRNAs with only 16 nts of complementarity showed significantly decreased or undetectable activities on the two sites for which they could be made (FIG. 3A). For each of the different sites tested, we transfected the same amounts of the full-length or shortened gRNA expression plasmid and Cas9 expression plasmid. Control experiments in which we varied the amounts of Cas9 and truncated gRNA expression plasmids transfected for EGFP sites #1, #2, and #3 suggested that shortened gRNAs function equivalently to their full-length counterparts (FIGS. 3E (bottom) and 3F (bottom)) and that therefore we could use the same amounts of plasmids when making comparisons at any given target site. Taken together, these results provide evidence that shortened gRNAs bearing 17 or 18 nts of complementarity can generally function as efficiently as full-length gRNAs and hereafter the truncated gRNAs with these complementarity lengths are referred to as “tru-gRNAs” and RGNs using these tru-gRNAs as “tru-RGNs”.

Whether tru-RGNs could efficiently induce indels on chromatinized endogenous gene targets was tested next. Tru-gRNAs were constructed for seven sites in three endogenous human genes (VEGFA, EMX1, and CLTA), including four sites that had previously been targeted with standard full-length gRNAs in three endogenous human genes: VEGFA site 1, VEGFA site 3, EMX1, and CTLA (Example 1 and Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013) (FIG. 3B). (It was not possible to test a tru-gRNA for VEGFA site 2 from Example 1, because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for gRNA expression from a U6 promoter.) Using a well-established T7 Endonuclease I (T7EI) genotyping assay (Reyon et al., 2012) as described above, the Cas9-mediated indel mutation frequencies induced by each of these various gRNAs at their respective target sites was quantified in human U2OS.EGFP cells. For all five of the seven four sites, tru-RGNs robustly induced indel mutations with efficiencies comparable to those mediated by matched standard RGNs (FIG. 3B). For the two sites on which tru-RGNs showed lower activities than their full-length counterparts, we note that the absolute rates of mutagenesis were still high (means of 13.3% and 16.6%) at levels that would be useful for most applications. Sanger sequencing for three of these target sites (VEGFA sites 1 and 3 and EMX1) confirmed that indels induced by tru-RGNs originate at the expected site of cleavage and that these mutations are essentially indistinguishable from those induced with standard RGNs (FIG. 3C and FIGS. 7A-D).

We also found that tru-gRNAs bearing a mismatched 5′ G and an 18 nt complementarity region could efficiently direct Cas9-induced indels whereas those bearing a mismatched 5′ G and a 17 nt complementarity region showed lower or undetectable activities compared with matched full-length gRNAs (FIG. 7E), consistent with our findings that a minimum of 17 nts of complementarity is required for efficient RGN activity.

To further assess the genome-editing capabilities of tru-RGNs, their abilities to induce precise sequence alterations via HDR with ssODN donor templates were tested. Previous studies have shown that Cas9-induced breaks can stimulate the introduction of sequence from a homologous ssODN donor into an endogenous locus in human cells (Cong et al., 2013; Mali et al., 2013c; Ran et al., 2013; Yang et al., 2013). Therefore, the abilities were compared of matched full-length and tru-gRNAs targeted to VEGFA site 1 and to the EMX1 site to introduce a BamHI restriction site encoded on homologous ssODNs into these endogenous genes. At both sites, tru-RGNs mediated introduction of the BamHI site with efficiencies comparable to those seen with standard RGNs harboring their full-length gRNA counterparts (FIG. 3D). Taken together, this data demonstrate that tru-RGNs can function as efficiently as standard RGNs to direct both indels and precise HDR-mediated genome editing events in human cells.

Example 2b Tru-RGNs Exhibit Enhanced Sensitivities to gRNA/DNA Interface Mismatches

Having established that tru-RGNs can function efficiently to induce on-target genome editing alterations, whether these nucleases would show greater sensitivity to mismatches at the gRNA/DNA interface was tested. To assess this, a systematic series of variants was constructed for the tru-gRNAs that were previously tested on EGFP sites #1, #2, and #3 (FIG. 3A above). The variant gRNAs harbor single Watson-Crick substitutions at each position within the complementarity region (with the exception of the 5′ G required for expression from the U6 promoter) (FIG. 5A). The human cell-based EGFP disruption assay was used to assess the relative abilities of these variant tru-gRNAs and an analogous set of matched variant full-length gRNAs made to the same three sites as described in Example 1 to direct Cas9-mediated indels. The results show that for all three EGFP target sites, tru-RGNs generally showed greater sensitivities to single mismatches than standard RGNs harboring matched full-length gRNAs (compare bottom and top panels of FIG. 5A). The magnitude of sensitivity varied by site, with the greatest differences observed for sites #2 and #3, whose tru-gRNAs harbored 17 nts of complementarity.

Encouraged by the increased sensitivity of tru-RGNs to single nucleotide mismatches, we next sought to examine the effects of systematically mismatching two adjacent positions at the gRNA-DNA interface. We therefore made variants of the tru-gRNAs targeted to EGFP target sites #1, #2, and #3, each bearing Watson-Crick transversion substitutions at two adjacent nucleotide positions (FIG. 5B). As judged by the EGFP disruption assay, the effects of adjacent double mismatches on RGN activity were again substantially greater for tru-gRNAs than for the analogous variants made in Example 1 for matched full-length gRNAs targeted to all three EGFP target sites (compare bottom to top panels in FIG. 5B). These effects appeared to be site-dependent with nearly all of the double-mismatched tru-gRNAs for EGFP sites #2 and #3 failing to show an increase in EGFP disruption activities relative to a control gRNA lacking a complementarity region and with only three of the mismatched tru-gRNA variants for EGFP site #1 showing any residual activities (FIG. 5B). In addition, although double mutations generally showed greater effects on the 5′ end with full-length gRNAs, this effect was not observed with tru-gRNAs. Taken together, our data suggest that tru-gRNAs exhibit greater sensitivities than full-length gRNAs to single and double Watson-Crick transversion mismatches at the gRNA-DNA interface.

Example 2c Tru-RGNs Targeted to Endogenous Genes Show Improved Specificities in Human Cells

The next experiments were performed to determine whether tru-RGNs might show reduced genomic off-target effects in human cells relative to standard RGNs harboring full-length gRNA counterparts. We examined matched full-length and tru-gRNAs targeted to VEGFA site 1, VEGFA site 3, and EMX1 site 1 (described in FIG. 3B above) because previous studies (see Example 1 and Fu et al., 2013; Hsu et al., 2013) had defined 13 bona fide off-target sites for the full-length gRNAs targeted to these sites. (We were unable to test a tru-gRNA for VEGFA site 2 from our original study6 because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for efficient gRNA expression from a U6 promoter.) Strikingly, we found that tru-RGNs showed substantially reduced mutagenesis activity in human U2OS.EGFP cells relative to matched standard RGNs at all 13 of these bona fide off-target sites as judged by T7EI assay (Table 3A); for 11 of the 13 off-target sites, the mutation frequency with tru-RGNs dropped below the reliable detection limit of the T7EI assay (2-5%) (Table 3A). We observed similar results when these matched pairs of standard and tru-RGNs were tested at the same 13 off-target sites in another human cell line (FT-HEK293 cells) (Table 3A).

To quantify the magnitude of specificity improvement observed with tru-RGNs, we measured off-target mutation frequencies using high-throughput sequencing, which provides a more sensitive method for detecting and quantifying low frequency mutations than the T7EI assay. We assessed a subset of 12 of the 13 bona fide off-target sites for which we had seen decreased mutation rates with tru-gRNAs by T7EI assay (for technical reasons, we were unable to amplify the required shorter amplicon for one of the sites) and also examined an additional off-target site for EMX1 site 1 that had been identified by another group7 (FIG. 6A). For all 13 off-target sites we tested, tru-RGNs showed substantially decreased absolute frequencies of mutagenesis relative to matched standard RGNs (FIG. 6A and Table 3B) and yielded improvements in specificity of as much as ˜5000-fold or more relative to their standard RGN counterparts (FIG. 6B). For two off-target sites (OT1-4 and OT1-11), it was difficult to quantify the on-target to off-target ratios for tru-RGNs because the absolute number and frequency of indel mutations induced by tru-RGNs fell to background or near-background levels. Thus, the ratio of on-target to off-target rates would calculate to be infinite in these cases. To address this, we instead identified the maximum likely indel frequency with a 95% confidence level for these sites and then used this conservative estimate to calculate the minimum likely magnitude of specificity improvement for tru-RGNs relative to standard RGNs for these off-targets. These calculations suggest tru-RGNs yield improvements of ˜10,000-fold or more at these sites (FIG. 6B).

To further explore the specificity of tru-RGNs, we examined their abilities to induce off-target mutations at additional closely related sites in the human genome. For the tru-gRNAs to VEGFA site 1 and EMX1, which each possess 18 nts of target site complementarity, we computationally identified all additional sites in the human genome mismatched at one or two positions within the complementarity region (not already examined above in Table 3A) and a subset of all sites mismatched at three positions among which we favored mismatches in the 5′ end of the site as described in Example 1. For the tru-gRNA to VEGFA site 3, which possesses 17 nts of target site complementarity, we identified all sites mismatched at one position and a subset of all sites mismatched at two positions among which mismatches in the 5′ end were favored (again not already examined in Table 3A). This computational analysis yielded a total of 30, 30, and 34 additional potential off-target sites for the tru-RGNs targeted to VEGFA site 1, VEFGA site 3, and the EMX1 site, respectively, which we then assessed for mutations using T7EI assay in human U2OS.EGFP and HEK293 cells in which the RGNs had been expressed.

Strikingly, the three tru-RGNs to VEGFA site 1, VEFGA site 3, and EMX1 did not induce detectable Cas9-mediated indel mutations at 93 of the 94 potential off-target sites examined in human U2OS.EGFP cells or at any of the 94 potential off-target sites in human HEK293 cells (Table 3C). For the one site at which off-target mutations were seen, whether the standard RGN with a full-length gRNA targeted to VEGFA site 1 could also mutagenize this same off-target site was examined; it induced detectable mutations albeit at a slightly lower frequency (FIG. 6C). The lack of improvement observed with shortening of the gRNA at this off-target site can be understood by comparing the 20 and 18 nt sequences for the full-length and tru-gRNAs, which shows that the two additional bases in the full-length 20 nt target are both mismatched (FIG. 6C). In summary, based on this survey of 94 additional potential off-target sites, shortening of the gRNA does not appear to induce new high-frequency off-target mutations.

Deep sequencing of a subset of the 30 most closely matched potential off-target sites from this set of 94 site (i.e.—those with one or two mismatches) showed either undetectable or very low rates of indel mutations (Table 3D) comparable to what we observed at other previously identified off-target sites (Table 3B). We conclude that tru-RGNs generally appear to induce either very low or undetectable levels of mutations at sites that differ by one or two mismatches from the on-target site. This contrasts with standard RGNs for which it was relatively easy to find high-frequency off-target mutations at sites that differed by as many as five mismatches (see Example 1).

TABLE 3A On- and off-target mutation frequencies of matched tru-RGNs and standard RGNs targeted to endogenous genes in human U2OS.EGFP and HEK 293 cells Indel mutation Indel mutation frequency frequency Tar- SEQ (%) ± s.e.m. SEQ (%) ± s.e.m. get ID U2OS. ID U2OS. ID 20 mer Target NO: EGFP HEK293 Truncated Target NO: EGFP HEK293 Gene T1 GGGTGGGGGGAGTTTGCTCCtGG 2242. 23.69 ±  6.96 ± GTGGGGGGAGTTTGCTCCtGG 2243. 23.93 ±  8.34 ± VEGFA  1.99  1.33  4.37  0.01 OT1- GG A TGG A GGGAGTTTGCTCCtGG 2244. 17.25 ±  7.26 ± A TGG A GGGAGTTTGCTCCtGG 2245. N.D. N.D. IGDCC3 3  2.97  0.62 OT1- GGG A GGG T GGAGTTTGCTCCtGG 2246.  6.23 ±  2.66 ± G A GGG T GGAGTTTGCTCCtGG 2247. N.D. N.D. LOC116437 4  0.20  0.30 OT1- C GG G GG A GGGAGTTTGCTCCtGG 2248.  3.73 ±  1.41 ± G G GG A GGGAGTTTGCTCCtGG 2249. N.D. N.D. CACNA2D 6  0.23  0.07 OT1- GGG GA GGGG A AGTTTGCTCCtGG 2250. 10.4 ±  3.61 ± G GA GGGG A AGTTTGCTCCtGG 2251. N.D. N.D. 11  0.7  0.02 T3 GGTGAGTGAGTGTGTGCGTGtGG 2252. 54.08 ± 22.97 ± GAGTGAGTGTGTGCGTGtGG 2253. 50.49 ± 20.05 ± VEGFA  1.02  0.17  1.25  0.01 OT3- GGTGAGTGAGTGTGTG T GTGaGG 2254.  6.16 ±  6.02 ± GAGTGAGTGTGTG T GTGaGG 2255. N.D. N.D. (abParts) 1  0.98  0.11 OT3- A GTGAGTGAGTGTGTG T GTGgGG 2256. 19.64 ± 11.29 ± GAGTGAGTGTGTG T GTGgGG 2257.  5.52 ±  3.41 ± MAX 2  1.06  0.27  0.25  0.07 OT3- G C TGAGTGAGTGT A TGCGTGtGG 2258.  7.95 ±  4.50 ± GAGTGAGTGT A TGCGTGtGG 2259.  1.69 ±  1.27 ± 4  0.11  0.02  0.26  0.10 OT3- GGTGAGTGAGTG C GTGCG G GtGG 2260. N.D.  1.09 ± GAGTGAGTG C GTGCG G GtGG 2261. N.D. N.D. TPCN2 9  0.17 OT3- G T TGAGTGA A TGTGTGCGTGaGG 2262.  1.85 ± N.D. GAGTGA A TGTGTGCGTGaGG 2263. N.D. N.D. SLIT1 17  0.08 OT3- T GTG G GTGAGTGTGTGCGTGaGG 2264.  6.16 ±  6.27 ± G G GTGAGTGTGTGCGTGaGG 2265. N.D. N.D. COMDA 18  0.56  0.09 OT3- A G A GAGTGAGTGTGTGC A TGaGG 2266. 10.47 ±  4.38 ± GAGTGAGTGTGTGC A TGaGG 2267. N.D. N.D. 20  1.08  0.58 T4 GAGTCCGAGCAGAAGAAGAAgGG 2268. 41.56 ± 12.65 ± GTCCGAGCAGAAGAAGAAgGG 2269. 43.01 ± 17.25 ± EMX1  0.20  0.31  0.87  0.64 OT4- GAGT TA GAGCAGAAGAAGAAaGG 2270. 19.26 ±  4.14 ± GT TA GAGCAGAAGAAGAAaGG 2271. N.D. N.D. HCN1 2  0.73  0.66 OT-4_ GAGTC TA AGCAGAAGAAGAAg A G 2272.  4.37 ± N.D. GTC TA AGCAGAAGAAGAAg A G 2273. N.D. N.D. MFAP1 Hsu31  0.58 Mutation frequencies were measured by T7EI assay. Means of duplicate measurements are shown with error bars representing standard errors of the mean. *Off-target site OT4_53 is the same as EMX1 target 3 OT31 from Hsu et al., 2013.

TABLE 3B Numbers of wild-type (WT) and indel mutation sequencing reads from deep sequencing experiments Control tru-RGN Standard RGN Site Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 45 140169 0.03% 122858 242127 33.66% 150652 410479 26.85% OT1-3 0 132152 0.00% 1595 205878 0.77% 50973 144895 26.02% OT1-4 0 133508 0.00% 0 223881 0.00% 22385 240873 8.50% OT1-6 3 213642 0.00% 339 393124 0.09% 24332 424458 5.21% OT1-11 1 930894 0.00% 0 274779 0.00% 43738 212212 17.09% VEGFA site 3 5 212571 0.00% 303913 292413 50.96% 183626 174740 51.24% OT3-2 1169 162545 0.71% 9415 277616 3.28% 26545 222482 10.66% OT3-4 7 383006 0.00% 15551 1135673 1.35% 42699 546203 7.25% OT3-9 73 145367 0.05% 113 227874 0.05% 1923 168293 1.13% OT3-17 8 460498 0.00% 31 1271276 0.00% 16760 675708 2.42% OT3-18 7 373571 0.00% 284 1275982 0.02% 72354 599030 10.78% OT3-20 5 140848 0.00% 593 325162 0.18% 30486 202733 13.07% EMX1 site 1 1 158838 0.00% 49104 102805 32.32% 128307 307584 29.44% OT4-1 10 169476 0.01% 13 234039 0.01% 47426 125683 27.40% OT4-52 2 75156 0.00% 10 231090 0.00% 429 340201 0.13% OT4-53 0 234069 0.00% 6 367811 0.00% 17421 351667 4.72% Freq. = frequency of indel mutations = number of indel sequences/number of wild-type sequences. Control gRNA = gRNA lacking a complementarity region

TABLE 3C Indel mutation frequencies at potential off-target sites of tru-RGNs targeted to endogenous genes in human cells Indel mutation frequency SEQ (%) ± s.e.m. ID Number of U2OS.EGFP Target ID Target Site + PAM NO: mismatches cells HEK293 cells VEGFA GTGGGGGGAGTTTGCTCCtGG 2274. 0 (on-target) 23.93 ± 4.37  8.34 ± 0.01 Site 1 GTGGGGGGAGTTTGC C CCaGG 2275. 1 Not detected Not detected GTGGGGGG T GTTTGCTCCcGG 2276. 1 Not detected Not detected GTGGG T GGAGTTTGCT A CtGG 2277. 2 Not detected Not detected GTGGGGGGAG C TT T CTCCtGG 2278. 2 Not detected Not detected GTGGG T GG C GTTTGCTCCaGG 2279. 2 Not detected Not detected GTGG A GGGAG C TTGCTCCtGG 2280. 2  6.88 ± 0.19 Not detected GTGGG T GGAGTTTGCT A CaGG 2281. 2 Not detected Not detected G G GGGGG C AGTTTGCTCCtGG 2282. 2 Not detected Not detected GTG T GGGGA A TTTGCTCCaGG 2283. 2 Not detected Not detected C TG CT GGGAGTTTGCTCCtGG 2284. 3 Not detected Not detected T T T GGG A GAGTTTGCTCCaGG 2285. 3 Not detected Not detected C TG A GGG C AGTTTGCTCCaGG 2286. 3 Not detected Not detected GT AA GGG A AGTTTGCTCCtGG 2287. 3 Not detected Not detected G G GGG TA GAGTTTGCTCCaGG 2288. 3 Not detected Not detected G G G T GGGGA C TTTGCTCCaGG 2289. 3 Not detected Not detected G G GGG A G C AGTTTGCTCCaGG 2290. 3 Not detected Not detected T TGGGG TT AGTTTGCTCCtGG 2291. 3 Not detected Not detected T TG A GGGGAGT C TGCTCCaGG 2292. 3 Not detected Not detected C TGGGG T GA T TTTGCTCCtGG 2293. 3 Not detected Not detected G A G A GGGGAGTT G GCTCCtGG 2294. 3 Not detected Not detected T T T GGGGGAGTTTGC C CCaGG 2295. 3 Not detected Not detected T T C GGGGGAGTTTGC G CCgGG 2296. 3 Not detected Not detected C T C GGGGGAGTTTGC A CCaGG 2297. 3 Not detected Not detected GTG TT GGGAGT C TGCTCCaGG 2298. 3 Not detected Not detected G A GGGGG C AG G TTGCTCCaGG 2299. 3 Not detected Not detected G A GGGG A GAGTTTG T TCCaGG 2300. 3 Not detected Not detected GTGG CT GGAGTTTGCT G CtGG 2301. 3 Not detected Not detected GT C GGGGGAGT GG GCTCCaGG 2302. 3 Not detected Not detected G A GGGGGGAGT G TG T TCCgGG 2303. 3 Not detected Not detected GTGG T GGGAG C TTG T TCCtGG 2304. 3 Not detected Not detected GTGGGGGG T G CC TGCTCCaGG 2305. 3 Not detected Not detected VEGFA GAGTGAGTGTGTGCGTGtGG 2306. 0 (on-target) 50.49 ± 1.25 20.05 ± 0.01 Site 3 C AGTGAGTGTGTGCGTGtGG 2307. 1 Not detected Not detected G T GTGAGTGTGTGCGTGgGG 2308. 1 Not detected Not detected G T GTGAGTGTGTGCGTGaGG 2309. 1 Not detected Not detected G T GTGAGTGTGTGCGTGtGG 2310. 1 Not detected Not detected GAGTG T GTGTGTGCGTGtGG 2311. 1 Not detected Not detected GAGTG G GTGTGTGCGTGgGG 2312. 1 Not detected Not detected GAGTGA C TGTGTGCGTGtGG 2313. 1 Not detected Not detected GAGTGAGTGTGTG G GTGgGG 2314. 1 Not detected Not detected GAGTGAGTGTGTG T GTGtGG 2315. 1 Not detected Not detected GAGTGAGTGTGTG T GTGtGG 2316. 1 Not detected Not detected GAGTGAGTGTGTG T GTGgGG 2317. 1 Not detected Not detected GAGTGAGTGTGTG T GTGtGG 2318. 1 Not detected Not detected GAGTGAGTGTGTGCG C GgGG 2319. 1 Not detected Not detected CT GTGAGTGTGTGCGTGaGG 2320. 2 Not detected Not detected AT GTGAGTGTGTGCGTGtGG 2321. 2 Not detected Not detected G CC TGAGTGTGTGCGTGtGG 2322. 2 Not detected Not detected G T GTG T GTGTGTGCGTGtGG 2323. 2 Not detected Not detected G T GTG G GTGTGTGCGTGtGG 2324. 2 Not detected Not detected G C GTG T GTGTGTGCGTGtGG 2325. 2 Not detected Not detected G T GTG T GTGTGTGCGTGgGG 2326. 2 Not detected Not detected G T GTG C GTGTGTGCGTGtGG 2327. 2 Not detected Not detected G T GTG T GTGTGTGCGTGcGG 2328. 2 Not detected Not detected GAG A GAG A GTGTGCGTGtGG 2329. 2 Not detected Not detected GAGTG T GTG A GTGCGTGgGG 2330. 2 Not detected Not detected G T GTGAGTGTGTG T GTGtGG 2331. 2 Not detected Not detected GAGTG T GTGT A TGCGTGtGG 2332. 2 Not detected Not detected GAGT C AGTGTGTG A GTGaGG 2333. 2 Not detected Not detected GAGTG T GTGTGTG A GTGtGG 2334. 2 Not detected Not detected GAGTG T GTGTGTGC A TGtGG 2335. 2 Not detected Not detected GAGTGAG A GTGTG T GTGtGG 2336. 2 Not detected Not detected GAGTGAGTG A GTG A GTGaGG 2337. 2 Not detected Not detected EMX1 site GTCCGAGCAGAAGAAGAAgGG 2338. 0 (on-target) 43.01 ± 0.87 17.25 ± 0.64 GTC T GAGCAGAAGAAGAAtGG 2339. 1 Not detected Not detected GTCC C AGCAG T AGAAGAAtGG 2340. 2 Not detected Not detected GTCCGAG G AGA G GAAGAAaGG 2341. 2 Not detected Not detected GTC A GAG G AGAAGAAGAAgGG 2342. 2 Not detected Not detected G A C A GAGCAGAAGAAGAAgGG 2343. 2 Not detected Not detected GT GG GAGCAGAAGAAGAAgGG 2344. 2 Not detected Not detected GT A C T AGCAGAAGAAGAAaGG 2345. 2 Not detected Not detected GTC T GAGCA C AAGAAGAAtGG 2346. 2 Not detected Not detected GT G C T AGCAGAAGAAGAAgGG 2347. 2 Not detected Not detected TA C A GAGCAGAAGAAGAAtGG 2348. 3 Not detected Not detected TA C G GAGCAGAAGAAGAAtGG 2349. 3 Not detected Not detected AA C G GAGCAGAAGAAGAAaGG 2350. 3 Not detected Not detected G A C AC AGCAGAAGAAGAAgGG 2351. 3 Not detected Not detected C T G CGA T CAGAAGAAGAAaGG 2352. 3 Not detected Not detected G A C T G G GCAGAAGAAGAAgGG 2353. 3 Not detected Not detected T TCC CT GCAGAAGAAGAAaGG 2354. 3 Not detected Not detected T TCC T A C CAGAAGAAGAAtGG 2355. 3 Not detected Not detected C TC T GAG G AGAAGAAGAAaGG 2356. 3 Not detected Not detected A TCC A A T CAGAAGAAGAAgGG 2357. 3 Not detected Not detected G C CC CT GCAGAAGAAGAAcGG 2358. 3 Not detected Not detected A TCC A A C CAGAAGAAGAAaGG 2359. 3 Not detected Not detected G A C T GAG A AGAAGAAGAAaGG 2360. 3 Not detected Not detected GT GG GA T CAGAAGAAGAAaGG 2361. 3 Not detected Not detected G A C A GAG A AGAAGAAGAAaGG 2362. 3 Not detected Not detected GTC ATG GCAGAAGAAGAAaGG 2363. 3 Not detected Not detected GT TG GAG A AGAAGAAGAAgGG 2364. 3 Not detected Not detected GT AA GAG A AGAAGAAGAAgGG 2365. 3 Not detected Not detected C TCC T AGCA A AAGAAGAAtGG 2366. 3 Not detected Not detected T TC A GAGCAG G AGAAGAAtGG 2367. 3 Not detected Not detected GT TG GAGCAG G AGAAGAAgGG 2368. 3 Not detected Not detected G C C T GAGCAGAAG G AGAAgGG 2369. 3 Not detected Not detected GTC T GAG G A C AAGAAGAAtGG 2370. 3 Not detected Not detected GTCCG G G A AG G AGAAGAAaGG 2371. 3 Not detected Not detected G G CCGAGCAGAAGAA AG AcGG 2372. 3 Not detected Not detected GTCC T AGCAG G AGAAGAAg A G 2373. 3 Not detected Not detected

TABLE 3D Frequencies of tru-RGN-induced indel mutations at potential off- target sites in human U2OS.EGFP as determined by deep sequencing On- target tru-RGN Control site Off-target site sequence S# Indel WT Freq. Indel WT Freq VEGFA GTGGGGGGAGTTTGC C CCaGG 2374. 1500 225640 0.66% 3 135451 0.00% site 1 GTGGGGGG T GTTTGCTCCcGG 2375. 1552 152386 1.01% 0 86206 0.00% GTGGG T GGAGTTTGCT A CtGG 2376. 1 471818 0.00% 0 199581 0.00% GTGGG T GGAGTTTGCT A CaGG 2377. 0 337298 0.00% 1 211547 0.00% GTGGG T GG C GTTTGCTCCaGG 2378. 2 210174 0.00% 1 105531 0.00% GTG T GGGGA A TTTGCTCCaGG 2379. 673 715547 0.09% 1 387097 0.00% GTGGGGGGAG C TT T CTCCtGG 2380. 5 107757 0.00% 1 58735 0.00% G G GGGGG C AGTTTGCTCCtGG 2381. 1914 566548 0.34% 3 297083 0.00% VEGFA G T GTGAGTGTGTGCGTGtGG 2382. 58 324881 0.02% 9 122216 0.01% site 3 G T GTGAGTGTGTGCGTGaGG 2383. 532 194914 0.27% 11 73644 0.01% GAGTG G GTGTGTGCGTGgGG 2384. 70 237029 0.03% 10 178258 0.01% GAGTGA C TGTGTGCGTGtGG 2385. 6 391894 0.00% 0 239460 0.00% GAGTGAGTGTGTG G GTGgGG 2386. 15 160140 0.01% 10 123324 0.01% G T GTGAGTGTGTGCGTGgGG 2387. 19 138687 0.01% 1 196271 0.00% C AGTGAGTGTGTGCGTGtGG 2388. 78 546865 0.01% 41 355953 0.01% G T GTGAGTGTGTGCGTGtGG 2389. 128 377451 0.03% 56 133978 0.04% GAGTG T GTGTGTGCGTGtGG 2390. 913 263028 0.35% 78 178979 0.04% GAGTGAGTGTGTG T GTGtGG 2391. 40 106933 0.04% 36 58812 0.06% GAGTGAGTGTGTG T GTGtGG 2392. 681  762999 0.09% 63 222451 0.03% GAGTGAGTGTGTG T GTGgGG 2393. 331 220289 0.15% 100 113911 0.09% GAGTGAGTGTGTG T GTGtGG 2394. 0 35725 0.00% 8 186495 0.00% GAGTGAGTGTGTGCG C GgGG 2395. 94 246893 0.04% 16 107623 0.01% EMX1 GTC A GAG G AGAAGAAGAAgGG 2396. 0 201483 0.00% 4 148416 0.00% site 1 GTC A GAG G AGAAGAAGAAgGG 2397. 10 545662 0.00% 5 390884 0.00% GTC T GAGCA C AAGAAGAAtGG 2398. 2 274212 0.00% 0 193837 0.00% GTC T GAGCAGAAGAAGAAtGG 2399. 440 375646 0.12% 10 256181 0.00% G A C A GAGCAGAAGAAGAAgGG 2400. 2 212472 0.00% 1 158860 0.00% GT A C T AGCAGAAGAAGAAaGG 2401. 152 229209 0.07% 103 157717 0.07% GT GG GAGCAGAAGAAGAAgGG 2402. 50 207401 0.02% 36 111183 0.03% GTCC C AGCAG T AGAAGAAtGG 2403. 0 226477 0.00% 1 278948 0.00% S#: SEQ ID NO:

Example 2d Tru-gRNAs can be Used with Dual Cas9 Nickases to Efficiently Induce Genome Editing in Human Cells

tru-gRNAs were tested with the recently described dual Cas9 nickase approach to induce indel mutations. To do this, the Cas9-D10A nickase together with two full-length gRNAs targeted to sites in the human VEGFA gene (VEGFA site 1 and an additional sequence we refer to as VEGFA site 4) were co-expressed in U2OS.EGFP cells (FIG. 4A). As described previously (Ran et al., 2013), this pair of nickases functioned cooperatively to induce high rates of indel mutations at the VEGFA target locus (FIG. 4B). Interestingly, Cas9-D10A nickase co-expressed with only the gRNA targeted to VEGFA site 4 also induced indel mutations at a high frequency, albeit at a rate somewhat lower than that observed with the paired full-length gRNAs (FIG. 4B). Importantly, use of a tru-gRNA for VEGFA site 1 in place of a full-length gRNA did not affect the efficacy of the dual nickase approach to induce indel mutations (FIG. 4B).

The dual nickase strategy has also been used to stimulate the introduction of specific sequence changes using ssODNs (Mali et al., 2013a; Ran et al., 2013) and so whether tru-gRNAs might be used for this type of alteration was also tested. Paired full-length gRNAs for VEGFA sites 1 and 4 together with Cas9-D10A nickase cooperatively enhanced efficient introduction of a short insertion from a ssODN donor (FIG. 3A) into the VEGFA locus in human U2OS.EGFP cells as expected (FIG. 3C). Again, the efficiency of ssODN-mediated sequence alteration by dual nicking remained equally high with the use of a tru-gRNA in place of the full-length gRNA targeted to VEGFA site 1 (FIG. 3C). Taken together, these results demonstrate that tru-gRNAs can be utilized as part of a dual Cas9 nickase strategy to induce both indel mutations and ssODN-mediated sequence changes, without compromising the efficiency of genome editing by this approach.

Having established that use of a tru-gRNA does not diminish the on-target genome editing activities of paired nickases, we next used deep sequencing to examine mutation frequencies at four previously identified bona fide off-target sites of the VEGFA site 1 gRNA. This analysis revealed that mutation rates dropped to essentially undetectable levels at all four of these off-target sites when using paired nickases with a tru-gRNA (Table 4). By contrast, neither a tru-RGN (Table 3B) nor the paired nickases with full-length gRNAs (Table 4) was able to completely eliminate off-target mutations at one of these four off-target sites (OT1-3). These results demonstrate that the use of tru-gRNAs can further reduce the off-target effects of paired Cas9 nickases (and vice versa) without compromising the efficiency of on-target genome editing.

TABLE 4 Frequencies of paired nickase-induced indel mutations at on- and off-target sites of VEGFA site 1 using full-length and tru-gRNAs Paired full-length gRNAs tru-gRNA/full-length gRNA Control Site Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 78905 345696 18.583% 65754 280720 18.978% 170 308478 0.055% OT1-3 184 85151 0.216% 0 78658 0.000% 2 107850 0.002% OT1-4 0 89209 0.000% 1 97010 0.001% 0 102135 0.000% OT1-6 2 226575 0.001% 0 208218 0.000% 0 254580 0.000% OT1-11 0 124729 0.000% 0 121581 0.000% 0 155173 0.000%

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Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A method of increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence.
 2. A method of inducing a break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell: a Cas9 nuclease or nickase; and a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of double-stranded DNA molecule.
 3. A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell: a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence.
 4. The method of claim 1, wherein the guide RNA is (i) a single guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, or (ii) a crRNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA.
 5. The method of claim 1, wherein the guide RNA is or comprises a ribonucleic acid selected from the group consisting of: (SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG (X_(N)), (SEQ ID NO: 2) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUC(X_(N)), (SEQ ID NO: 3) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAA GUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)), (SEQ ID NO: 4) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

wherein X₁₇₋₁₈ is a complementarity region that is complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), and X_(N) is any sequence, wherein N can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9.
 6. A guide RNA molecule having a target complementarity region of 17-18 nucleotides.
 7. The gRNA of claim 6, wherein the target complementarity region consists of 17-18 nucleotides.
 8. The gRNA of claim 6, wherein the target complementarity region consists of 17-18 nucleotides of target complementarity.
 9. The gRNA of claim 6, consisting of the sequence: (SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG (X_(N)), (SEQ ID NO: 2) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUC(X_(N)), (SEQ ID NO: 3) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAA GUUAAAAUAAGGCUAGUCCGUUAUC(X_(N)), (SEQ ID NO: 4) (X₁₇₋₁₈)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

wherein X₁₇₋₁₈ is a sequence complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), and X_(N) is any sequence, wherein N can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9.
 10. The method of claim 5, wherein the ribonucleic acid includes one or more U at the 3′ end of the molecule.
 11. The method of claim 5, wherein the ribonucleic acid includes one or more additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.
 12. The method of claim 5, wherein the ribonucleic acid includes one, two, or three additional nucleotides at the 5′ end of the RNA molecule that are not complementary to the target sequence.
 13. The method of claim 1, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence.
 14. The method of claim 1, wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target sequence.
 15. A DNA molecule encoding the ribonucleic acid of claim
 6. 16. A vector comprising the DNA molecule of claim
 15. 17. A host cell expressing the vector of claim
 16. 18. The method of claim 1, wherein the target region is in a target genomic sequence.
 19. The method of claim 1, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif (PAM).
 20. The method of claim 4, wherein the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) and the tracrRNA is GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA CUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8); the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404) and the tracrRNA is UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGC (SEQ ID NO:2405); or the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) and the tracrRNA is AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2406).
 21. The method of claim 3, wherein the dCas9-heterologous functional domain fusion protein (dCas9-HFD) comprises a HFD that modifies gene expression, histones, or DNA.
 22. The method of claim 21, wherein the heterologous functional domain is a transcriptional activation domain, an enzyme that catalyzes DNA demethylation, an enzyme that catalyzes histone modification, or a transcription silencing domain.
 23. The method of claim 22, wherein the transcriptional activation domain is from VP64 or NF-κB p65.
 24. The method of claim 22, wherein the enzyme that catalyzes histone modification is LSD1, a histone methyltransferase (HNMT), histone acetyltransferase (HAT), histone deacetylase (HDAC), or histone demethylase.
 25. The method of claim 22, wherein the transcription silencing domain is from Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β.
 26. The method of claim 1, which results in an indel mutation or sequence alteration in the selected target genomic sequence.
 27. The method of claim 1, wherein the cell is a eukaryotic cell.
 28. The method of claim 27, wherein the cell is a mammalian cell.
 29. The method of claim 4, wherein the tracrRNA consists of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA CUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8), or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGC (SEQ ID NO:2405) or an active portion thereof; AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof; CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof. 